UVSpectros

UV-VIS
SPECTROSCOPY
Khadeja Mahmud Al-masri.
Department of Physics, Al-Najah University
Experimental Physics 422595
Prof. Issam Ashqar
15 July 2020
spectrophotometer
ABSTRACT
 Spectroscopy is the study of interaction of electromagnetic radiation with matter. 1
 Basic principle of spectroscopy is the Beer-Lambert’s law.

 In UV-VIS Spectroscopy, a continuum range of wavelengths from 200nm to
900nm is used.2
 Spectrophotometer device is used in UV-VIS Spectroscopy. See figure1.

 Components of spectrophotometer
 Sources of light(200nm to 900nm).
 Monochromator.
 Sample solution in cuvette.
 Detectors.
 Readout devices. FIG 1.Spectophotometer.
 Types of spectrophotometer Single and double beam instruments.

 Applications of UV-VIS Spectroscopy Qualitative &Quantitative analyses. 3
INTRODUCTION
 Spectroscopy is the branch of science which deals with
interaction of electromagnetic radiation with materials. In other
words it is an analytical method for qualitative and quantitative
analysis by use of light.
 The Lambert Beer law in 1852 made the basis for the
quantitative evaluation of absorption measurements.2
 This led firstly to colorimetry, then to photometry and
finally to spectrophotometry.
 This evolution was along with the development of

detectors for measuring light intensities, i.e. silicon
photo-diode detector.
 Spectroscopy is widely used as an exploratory tool in the

fields of physics, , and chemistry, for determination of
composition, physical structure and electronic structure
of matter at atomic scale, molecular scale, and macro
scale.4
1.SPECTROSCOPY
 When an Electromagnetic radiation is incident on a

matter, phenomena like reflection, transmission,
absorption ,are occurring.4
 Spectroscopy is the study of interaction of

electromagnetic radiation with matter based on the Bohr-
Einstein frequency relationship E=hv , here h is the
proportionality constant called Planck’s constant (6.626
x 10-34 J s) and v is frequency.
 Measurement of radiation intensity as a function of wavelength
is described by spectroscopy, as shown in figure 2.
FIG.2.Spectroscopy graph.
 All forms of spectroscopy use part of the electromagnetic
radiation to give us information about the materials.
1.1 SPECTRUM
 The spectrum is formed by electromagnetic waves and
the wavelength is varies. See figure 3.
FIG.3.Electromagnatic spectrum.
 When a narrow beam of light is allowed to pass through
a prism/grating, it is dispersed into seven colors from red
to violet and the band is called
Spectrum..
See figure 4.
FIG.4.Glase prism dispersion

1.2 PRINCIPLE
Basic principle of spectroscopy is the Beer-Lambert’s law.2
1.2.1 BEER LAW

 Beer's law stated that absorbance is proportional to the
concentrations of the material sample.
1.2.2 LAMBERT LAW

 Lambert's law stated that absorbance of a material is directly
proportional to its thickness (path length).
 The modern derivation of the Beer–Lambert law combines the two
laws and correlates the absorbance to both the concentrations and the
thickness of the material.
FIG 5. Beer–Lambert law.

2. UV-VISIBLE SPECTROSCOPY
 Ultraviolet–visible spectrum can be generated when
ultraviolet light and visible light(200-900nm) are absorbed
by materials. The spectrum can be used to analyze the
composition and the structure of the materials. For a
particular wavelength in the ultraviolet–visible ranges, the
absorption degree is proportional to the components of the
materials. Therefore, the characteristics of the materials are
quantitatively reflected by the spectrum, which changes with
the wave-length.1
 Ultraviolet–visible spectrum consists of an absorption

spectrum. An absorption spectrum gives information about
the molar absorptivity, concentration of the sample, optical
bath length. See figure6, in previous slid.
2.1 INSTRUMENTATION
 2.1.1 SOURCE of LIGHT.
 2.1.2 MONOCHROMATOR.
 2.1.3 SMPLE SOLIOTION in CUVETTE.
 2.1.4 PHOTO DETECTOR.
 2.1.5 READOUT DEVICE.
FIG 6.Components of spectrophotometer.

2.1.1 SOURCE OF LIGHT
 Part of the UV and Visible radiation source is Tungsten lamp.

See figure 7.
FIG.7.Tungsten lamp
 UV radiation source is Deuterium or Hydrogen lamp . See
figure 8.
 Range of wavelength 200-400 nm.
FIG.8.Deuterium lamp
2.1.2 MONOCHROMATOR
 It is a device that breaks the polychromatic radiation into component
wavelengths. See figure 9.
FIG.9.Monochromator components.
The monochromator unit consists of :
•Entrance slit: defines narrow beam of radiation from source.
•Collimating mirror:(polished surface) collimates the lights.
•Diffraction grating or Prism (make of quartz): disperses the

light into specific wavelength.
•Focusing mirror: captures the dispersed light & sharpens the

same to the sample via exit slit
•Exit slit: allows the corrected wavelength of light to the

sample .
2.1.3 SMPLE SOLIOTION IN CUVETTE
 liquid sample is usually contained in a cell called a cuvette. See
figure10.
 Fingerprints and droplets of water disrupt light rays during
measurement.
 Cuvette from Quartz can be used in UV as well as in visible
spectroscopy.
 Cuvette from Glass is suitable for visible but not for UV spectroscopy
because it absorbs UV radiation.
FIG 10.sample solution in cuvette

2.1.4 PHOTO DETECTOR
 A photo detector is a semiconductor device which converts light
energy to electrical energy. It consists of a simple P-N junction diode
and is designed to work in reverse biased condition. The photons
approaching the diode are absorbed by the photodiode and current is
generated.4 See figure 11.
FIG 11. Photodiode

2.1.5 READOUT DEVICE.
 Digital screen to record an uv spectrograph with
absorbance against the wavelength.
------------------------------------------------
2.2 TYPES of SPECTROPHOTOMETER
FIG 12. Types of spectrophotometer.

2.3 APPLICATIONS OF UV-VIS
SPECTROSCOPY.
 UV-Vis spectroscopy is used heavily in many different research
areas to identify or quantify a sample.3
 Chemical field:
1. Detection of impurities.
2. Structure of organic compounds (single or double bond,

presence or absence of functional group).
3. Kinetics of reaction.
4. Manufacturing drugs.
 Biological fields
1. quantify the amount of protein and DNA in a sample
2. quantify the amount of bacterial cells in a cell culture

CONCLUSION
 Major advantages of uv-vis spectroscopy are:
1. High sensitivity.
2. Require only small volume of sample.
3. Linearity over wide range of concentration.
4. Can be used with gradient elution.4
 Major disadvantages of uv-vis spectroscopy are:

1. Not linear for high concentration.
2. Does not work with compounds that do not absorb light at
this wavelength region.
3. Generates significant heat and requires external cooling.4
REFERENCES
 1 H.H. Perkampus. UV-VIS Spectroscopy and its Applications,(2013),pp. 15-20.
 2 J.Q .Brown,and G.M .Palmer. Advances in quantitative UV–visible spectroscopy for

clinical and pre-clinical application in cancer,(2009), pp.30-42.
 3 M.M. Giusti, and R.E. Wrolstad. Characterization and measurement of anthocyanins by

UV‐visible spectroscopy,(2001),pp.57-60.
 4N.M.Kutty, UV-Visible spectroscopy, WWW Document (http://

reports.ias.ac.in/report/19397/uv-visible-spectroscopy)
ACKNOWLEDGEMENTS
 First of all, I am thankful to my guide, then to Dr. Issam Ashqar, who has guided me
throughout this course and made this project better.
QUESTIONS FOR
SELF-EVALUATION
 what is uv spectroscopy?
 What are the components of uv spectroscopy?
 What are the principles of uv spectroscopy?
 what are the applications of uv spectroscopy?

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UV-VIS

Experimental Physics 422595

Prof. Issam Ashqar

 Basic principle of spectroscopy is the Beer-Lambert’s law.

 Spectrophotometer device is used in UV-VIS Spectroscopy. See figure1.

 Types of spectrophotometer Single and double beam instruments.

 This evolution was along with the development of

 Spectroscopy is widely used as an exploratory tool in the

 When an Electromagnetic radiation is incident on a

 Spectroscopy is the study of interaction of

FIG.4.Glase prism dispersion

Basic principle of spectroscopy is the Beer-Lambert’s law.2

1.2.1 BEER LAW

1.2.2 LAMBERT LAW

FIG 5. Beer–Lambert law.

 Ultraviolet–visible spectrum consists of an absorption

 2.1.3 SMPLE SOLIOTION in CUVETTE.

 2.1.4 PHOTO DETECTOR.

 2.1.5 READOUT DEVICE.

FIG 6.Components of spectrophotometer.

 Part of the UV and Visible radiation source is Tungsten lamp.

•Entrance slit: defines narrow beam of radiation from source.

•Collimating mirror:(polished surface) collimates the lights.

•Diffraction grating or Prism (make of quartz): disperses the

•Focusing mirror: captures the dispersed light & sharpens the

•Exit slit: allows the corrected wavelength of light to the

FIG 10.sample solution in cuvette

FIG 11. Photodiode

FIG 12. Types of spectrophotometer.

2. Structure of organic compounds (single or double bond,

1. quantify the amount of protein and DNA in a sample

2. quantify the amount of bacterial cells in a cell culture

 Major disadvantages of uv-vis spectroscopy are:

 2 J.Q .Brown,and G.M .Palmer. Advances in quantitative UV–visible spectroscopy for

 3 M.M. Giusti, and R.E. Wrolstad. Characterization and measurement of anthocyanins by

 4N.M.Kutty, UV-Visible spectroscopy, WWW Document (http://

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