talks about spectrophotometer and its application

1. Spectrophotometry and its applications
Fundamentals of Computer and its Apolication
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May 6, 2022

2. Spectrophotomet
er A spectrophotometer is an instrument used to measure
wavelengths of electromagnetic radiation (light) that has interacted
with a sample [Nemcová, 1996].
Incident light can be reflected off, absorbed by, or transmitted through
a sample; the way the incident light changes during the interaction
with the sample is characteristic of the sample[Morris, 2015].
A spectrophotometer measures this change over a range of
incident wavelengths (or at a specific wavelength).
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3. Principl
e
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The fundamental theory is that light is absorbed or emitted over a
certain wavelength spectrum by each compound. The
spectrophotometer works by passing a light beam through a sample
to measure the light intensity of a sample [Kozioł et al., 2016].
Beer-Lambert Law It measures an amount of light that a
sample absorbs. It states that the amount of light absorbed is
directly proportional to the concentration of the solute in the
solution and thickness of the solution under
analysis.[Karpinska, 2012]
A = Ecl
▶ A is absorbance of the sample displayed on the
spectrophotometer
▶ E is Molar extinction coefficient
▶ c is concentration of the sample
▶ l is the path length

4. Instrumentatio
n
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A spectrophotometer is made up of two instruments: a spectrometer
and a photometer. The spectrometer is to produce light of any
wavelength, while the photometer is to measure the intensity of
light[Albert et al., 2012]. The spectrophotometer is designed in a
way that the liquid or a sample is placed between spectrometer and
photometer. The photometer measures the amount of light that
passes through the sample and delivers a voltage signal to the
display. If the absorbing of light change, the voltage signal also
changes
[Rogatkin et al., 2011]

5. Types of
Spectrophotometer
Types
Colorimete
r
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Features
Simple, more portable, less complex and Less versatile
than
spectrophotometers
Turbidometer measures the cloudiness or turbidity of a
solution
UV continuous or discontinuous spectral UV- light ranging
be- tween 200-450 nm, determines the absorbance or
transmit- tance for the fluids and even solutions
emitting a visible spectral range between 330-900 nm,
mea- sure the change in colour intensity according to
the change in the concentration of moderately diluted
solutions
are the most widely used. Visible spectrophotometers
are
a less expensive option if your applications are
colorimetric
Visibl
e
UV/Vis
Infrared
Single
beam
Doubl
e
makes the use of Nernst glowers as a conductive
device One sample is measured at a time. Single-
beam is the simplest and least expensive option
beam is split into two. One beam passes through the
sam- ple; the other passes through the reference
sample

6. Mechanis
m
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A spectrophotometer involve major events as described below
1. Firstly, a light source falls onto the monochromator
(Dispersion device).
2. Then, the monochromator will produce a single source of light
that falls onto the focusing wavelength selector.
3. The focusing convex lens will pass a fraction of the
monochromatic light source from the sample solution to the
photocell detector.
4. A photocell detector converts the light energy into electrical
energy, and an amplifier transmits this electrical signal to the
internalcircuit.
5. Finally, an internal circuit inside a spectrophotometer gives out
a final output on a digital meter.

7. Applications of
Spectrophotometer
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Following are the applications of spectrophotometer
▶ Quantification of compounds
▶ Quality Control: Contamination (purity ofsamples)
▶ Chemical Kinetics: monitor the progress of reaction (throughS/P)
▶ Detectors in liquid chromatography instruments
▶ Determination of melting temperature of DNA: Tm
▶ Microbial growth kinetics: (turbidometry/OD)
▶ Molecular weight determination of compounds
▶ Molecular weight determination of compounds
▶ Structure elucidation of organic compounds
▶ Monitoring dissolved oxygen content in freshwater and
marine ecosystems
▶ Detection of functional groups

8. Application
s
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9. Reference
s I Albert, D. R., Todt, M. A., and Davis, H. F. (2012).
A low-cost quantitative absorption
spectrophotometer. Journal of Chemical Education,
89(11):1432–1435.
Karpinska, J. (2012).
Basic principles and analytical application of
derivative spectrophotometry.
Macro to nano spectroscopy, book edited by Jamal Uddin,
pages
253–256.
Kozioł, M., Wotzka, D., Boczar, T., and Frącz, P. (2016).
Application of optical spectrophotometry for analysis of
radiation spectrum emitted by electric arc in the air.
Journal of Spectroscopy, 2016.
Morris, R. (2015).
Spectrophotometr
y.
Current Protocols Essential Laboratory Techniques, 11(1):2–
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10. References
II Nemcová, I. (1996).
Spectrophotometric reactions, volume
22. CRC Press.
Rogatkin, D., Sokolovski, S., Fedorova, K., Stewart, N., Sidorov,
V., and Rafailov, E. (2011).
Basic principles of design and functioning of multifunctional
laser diagnostic system for non-invasive medical
spectrophotometry.
In Advanced Biomedical and Clinical Diagnostic Systems IX,
volume
7890, pages 215–221. SPIE.
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