UV-Vis

Spectrophotometry
Antolihao, Christian
Buenaventura, Karshly Ira
 UV-Vis Spectrophotometry
Spectrophotometry is a quantitative measurement of the
absorption/transmission or reflection of a material as a function of wavelength.

UV-Vis Spectroscopy or Ultraviolet-Visible Spectrophotometry is a quantitative

technique used to measure how much a chemical substance absorbs light.

This is done by measuring the intensity of light that passes through a sample
with respect to the intensity of light through a reference sample or blank.

This means it uses light in the visible and adjacent ranges. The absorption or
reflectance in the visible range directly affects the perceived color of the
chemicals involved.
 UV-Vis Spectrophotometer
A UV-Vis spectrophotometer measures
the intensity of light transmitted through
a sample compared to a reference
measurement of the incident light
source.

Ultraviolet-Visible spectrophotometers
are able to determine the concentration
of specific analytes in a microvolume by
controlling the analysis wavelengths and
the pathlength.
 Uses of UV-Vis
Spectrophotometry
UV-Vis spectrometry is simple,
inexpensive and easy to use. It is
applicable for many different not too Environmental
complex samples. Therefore UV/Vis is
UV-Vis spectrometry is used as a
used in a broad range of areas, mainly
quick, first nonspecific screening
for routine measurements, for
of unwanted compounds in
example in hospitals, petrochemical
water. By measuring the color of
industry, food industry, water quality
process water during the
control laboratories. Also, universities,
production of drinking water we
chemical and biological plants use UV-
can determine if there are
Vis.
unwanted compounds in the
water.
 Uses of UV-Vis
Spectrophotometry
Pharmaceuticals
Vaccines
A vaccine is a type of treatment aimed at Qualitative analysis of organic compounds
stimulating the body’s immune system to fight can be achieved through the simple
against infectious pathogens such as bacteria process of UV-Vis spectrophotometry. UV-
and viruses. They are, according to the World Vis spectrophotometers measure the
Health Organization, “one of the most effective visible regions of ultraviolet light and can
ways to prevent disease”. provide valuable information about the
levels of active ingredients present in
UV Vis spectrophotometry provides fast, easy pharmaceutical compounds, as well as
and accurate characterization of components detect any impurities.
such as nucleic acids (i.e. DNA/RNA), proteins,
additives/preservatives, etc. and can impact
the time-to-result for both downstream and
upstream processes including quality control.
 Working Principle of a
UV-Vis Spectrophotometer
A spectrophotometer consists of three main components –
entrance slit, grating and detector.

-
Entrance Slit
Light from the source enters the entrance slit and the
size of the slit determines the amount of light that can
be measured by the instrument. The slit size also affects
the optical resolution of the spectrophotometer, where

-
the smaller the slit size, the better the resolution.
The beam becomes divergent after passing through the
slit and by reflecting the divergent beam on a
collimating mirror, the beam becomes collimated.
Collimated rays are then directed towards a diffraction
grating. The grating acts as a dispersive element and
splits the light into its constituent wavelengths.
 Working Principle of a
UV-Vis Spectrophotometer
-
Grating
A monochromator uses a phenomenon of optical
dispersion in a prism or diffraction from diffraction
gratings to select a particular wavelength of light. In
traditional spectrophotometers, prisms were used to

-
disperse light.
Once the light hits the diffraction grating, each
wavelength is reflected at a different angle. Diffraction
grating of different sizes are also used to determine

-
different wavelength ranges.
The beam becomes divergent again after being reflected
from the grating, thus it hits a second mirror to focus
and direct it towards the detector.
 Working Principle of a
UV-Vis Spectrophotometer

-
Detector
The detector captures the light spectra and measures
the intensity of light as a function of wavelength. These
data are then digitized and plotted onto a software as a
graph.
 Light Sources
Light sources commonly
found in a spectrophotometer
are made of Tungsten
Halogen, Deuterium, Xenon
Arcs, LED, Mercury Argon, Zinc
or Lasers.
Software
Much software can be implemented
for usage with spectrophotometers
for data acquisition. Most
companies producing the
instrument would also provide
software that is compatible with the
spectrometer they produce.
Interface
Most spectrophotometer systems
interface with the computer via
USB, RS-232 or Ethernet, newer
systems are able to transfer data
wirelessly using Wi-Fi and
Bluetooth.
Entrance Slit Mirrors
Slits come in a variety of sizes, from 5μm
The most common types of
to 800μm with a 1mm to 2mm height.
mirrors around are usually plane
The size of the slit depends on the
and spherical mirrors. However,
application and the most common slits
in a spectrophotometer, concave
used are in widths 10, 25, 50, 100 and
spherical mirrors are usually
200μm.
used.
Diffraction Gratings
Ruled gratings can be blazed for specific
Parts of a wavelengths and usually have a higher efficiency
UV-Vis than holographic gratings. Holographic gratings
Spectrophotometer tend to have a more uniform groove form and
spacing and generate less stray light as they are
produced optically.
Holders
Detectors
There is a wide variety of detectors being The samples are usually placed inside a
transparent cell, called cuvette. Test
used in different spectrophotometers and
some commonly used detectors are the tubes can also be used in place of
cuvettes in some equipment. The
photomultiplier tube (PMT), photodiode,
photodiode array, charge-coupled device material used to produce the cuvette
depends on the spectral range that the
(CCD), bolometer and multi-channel
analyzer (MCA). spectrophotometer covers.
 References
Hooijschuur, J. H. (n.d.). UV/VIS SPECTROMETRY BASICS. Retrieved from Chromedia Analytical Science:
www.chromedia.org/chromedia?waxtrapp=fotjtbEsHiemBpdmBlIEcCFpB&subNav=lnijabEsHiemBpdmBlIEcCFpBN
Phillips, K. (2015, March 13). UV Spectrophotometry Identifies Compounds in Pharmaceuticals. Retrieved from
HunterLab: blog.hunterlab.com/blog/color-pharmaceuticals/qualitative-analysis-how-uv-spectrophotometry-help-
identify-organic-compounds-in-pharmaceuticals/
Ultraviolet–visible spectroscopy. (2021, April 19). Retrieved from Wikipedia:
https://en.wikipedia.org/wiki/Ultraviolet%E2%80%93visible_spectroscopy
UV-Vis Spectroscopy. (n.d.). Retrieved from Edinburgh Instruments: https://www.edinst.com/us/techniques/uv-vis-
spectroscopy/
What is a UV-Vis Spectrophotometer? (2018, Oct. 16). Retrieved from DeNovix: https://www.denovix.com/blog/what-is-a-
uv-vis-spectrophotometer/
Xuan, N. C. (2020, APr. 29). What is a Spectrometer? UV, VIS, and IR Spectrometer Explained. Retrieved from wavelength-
oe.com: https://wavelength-oe.com/blog/what-is-a-spectrometer/
 Thank
you
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