Introduction Properties of electromagnetic radiation The electromagnetic spectrum & its usage for spectroscopic method UV visible spectrometry Principle Device and mechanism Applications Mass spectrometry Parts of mass spectrometer Theoretical example Creating ions IR spectrometry Theory Vibrational modes Raman spectrometry Theory Applications References

1. SPECTROMETRY
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By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. Synopsis
• Introduction
• Properties of electromagnetic radiation
• The electromagnetic spectrum & its usage for spectroscopic method
• UV visible spectrometry
 Principle
 Device and mechanism
 Applications
• Mass spectrometry
 Parts of mass spectrometer
 Theoretical example
 Creating ions
• IR spectrometry
 Theory
 Vibrational modes
• Raman spectrometry
 Theory
 Applications
• References
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3. Introduction
• Every chemical compound absorbs, transmits, or reflects light
(electromagnetic radiation) over a certain range of
wavelength.
• Spectrometry is a measurement of how much a chemical
substance absorbs or transmits.
• Spectrometry is widely used for quantitative analysis in
various areas (e.g., chemistry, physics, biology, biochemistry,
material and chemical engineering, clinical applications,
industrial applications, etc).
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4. Properties of electromagnetic radiation
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5. The electromagnetic spectrum and its
usage for spectroscopic method
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6. UV visible spectrometry
• Ultraviolet-visible spectrometry (UV-Vis or
UV/Vis) refers to absorption spectroscopy in
the ultraviolet-visible spectral region.
• 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.
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7. Devices and mechanism
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8. Transmittance(T) =
Absorbance(A)=−log(T)
= −log
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9. Beer-Lambert Law
We can determine the unknown concentration of
the sample by using Beer-Lambert Law.
Beer's Law is written as
A = ϵlc
• A is the measure of absorbance (no units),
• ϵ is the molar extinction coefficient or molar
absorptivity (or absorption coefficient),
• l is the path length, and
• c is the concentration.
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10. Applications of Ultraviolet – visible spectroscopy
• To determine concentration
• To identify unknown compound
• To asses quality of sample
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11. max ( maximum absorbance (in nm)
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12. If we know the optical density of following bio-molecules we can easily calculate the
concentration of these molecules which are shown in below table.
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13. Mass spectroscopy
• The mass spectrometer is an instrument that
used to calculate the mass of molecule.
• A mass spectrometer consists of three
components: an ion source, a mass analyzer,
and a detector.
• The ionizer converts a portion of the sample
into ions. (MALDI, ESI, FAB).
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14. basic structure of mass spectrometer
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15. Infrared spectroscopy
IR spectroscopy is a technique that uses
infrared electromagnetic radiation to
determine the functional groups that are
present in molecules.
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16. The infrared portion of the electromagnetic spectrum is
usually divided into three regions; the near-, mid- and
far- infrared
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17. Theory
• Just as electron can gain energy and transition
to excited states molecules can also transition
to higher energy level if they gain energy in
just the right amount.
• The bonds in molecules can be imagined to
vibrate with a certain frequency and energy
that is specific to that bond, just like masses
vibrate on springs.
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18. Modes of vibration
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19. Number of vibrational modes
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20. By using the table and graph determine
which molecule closely match the data
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21. Raman spectrometry
• Raman spectroscopy named after Indian
physicist Sir C. V. Raman) is a spectroscopic
technique used to observe vibrational,
rotational, and other low-frequency modes in
a system.
• Raman spectroscopy is commonly used in
chemistry to provide a structural fingerprint
by which molecules can be identified.
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22. Theory
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23. References
• K. Wilson and J. Walker: principle and
techniques of biotechnology and molecular
biotechnology. Seventh Edition.
• Spectrophotometry - Chemistry LibreTexts.
• Instrumental methods of chemical analysis
(Analytical Chemistry) By Dr. B. K. Sharma.
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