spectroscopy, classification of spectroscopy, history, UV-VIS spectrophotometer, principle, beer lambert law instrumentation, detector, single beam, double beam in time, double beam in space, application, merits, and demerits

1. ULTRAVIOLET-VISIBLE
SPECTROPHOTOMETRY
Presented By: GEETARANI LOUSHIGAM
Of MSc Food Technology and Quality Assurance, 2020-22
COLLEGE OF INDIGENEOUS FOOD TECHNOLOGY, KONNI
FOOD ANALYSIS AND INSTRUMENTATION

2. INTRODUCTION
1
UV-VIS SPECTROSCOPY
2
INSTRUMENTATION
3
CONCLUSION
4

3. Introduction
Spectroscopy is the study of the measurement and interpretation of electromagnet
ic radiation absorbed or emitted when the molecules or atoms or ions of a sample
moves from one energy state to another energy state.
An analyst carries out measurements of light (or light-induced charged particles)
that is absorbed, emitted, reflected or scattered by an analyte chemical or a
material. Then these measured data are correlated to identify and quantify the
chemical species present in that analyte.

4. 1. Atomic spectroscopy:
This kind of spectroscopy is concerned with the
interaction of electromagnetic radiation with
atoms which are commonly in their lowest
energy state, called the ground state.
2. Molecular spectroscopy:
This spectroscopy deals with the interaction of
electromagnetic radiation with molecules. The
interaction process results in a transition
between rotational and vibrational energy levels
in addition to electronic transitions.
Type Of Analytes type of radiation
Gamma-ray emission spectroscopy
X-Ray absorption/emission/fluorescence/diffraction
Vacuum ultraviolet absorption spectroscopy
Ultraviolet-visible absorption/emission/fluorescence
Infra-red absorption spectrophotometry
FT-IR spectroscopy
Raman scattering spectroscopy
Microwave absorption spectroscopy
Electron spin resonance spectroscopy
Nuclear magnetic resonance spectroscopy

5. UV-VIS SPECTROSCOPY
1941
1940
1852
The basis for the
quantitative evalua
tion of absorption
measurements.
The Lambert Beer
law
Beckman and
colleagues at
National Technologies
Laboratories.
First Lab
Spectrophotometer
Arnold Beckman introduced
the production version of the
Model D prototype that he
and Howard Cary had first
built.
DU UV-VIS
Spectrophotometer

6. Ultraviolet-visible spectroscopy is used to obtain the absorbance of light
energy or electromagnetic radiation, which excites electrons from the
ground state to the first singlet excited state of the compound or material.
The electromagnetic radiation (185 nm to 760 nm) that is provided by the
instrument is absorbed by the analyte, and the amount of absorption
is measured.
The absorption degree is proportional to the components of the materials.
Therefore, the characteristics of the materials are quantitatively reflected
by the spectrum.

7. PRINCIPLE
Credits is given to German physicist Johann
Lambert, although he really extended concepts
originally developed by French scientist Pierre
Bouguer (1729).
German scientist August Beer later applied si
milar experiments to solutions of different co
ncentrations and published his results
Lambert's law(1760) stated that absorbance of a m
aterial is directly proportional to its thickness (path
length).
(August 26, 1728 – September 25, 1777)
JOHANN HEINRICH LAMBERT
(31 July 1825 – 18 November 1863)
AUGUST BEER
PIERRE BOUGUER
(16 February 1698 – 15 August 1758)
Beer's law(1852) stated that absorbance is proporti
onal to the concentrations of the material sample.

8. When a beam of radiation (light) passes through a substance
or a solution, some of the light may be absorbed and the
remainder transmitted through the sample.
The absorbance of a sample is proportional to the absorptivity
of the substance (α), the path length(l) and the concentration
of the absorbing substance (c).
A = α * l * c
where α = the absorptivity of the substance
l = path length
c = concentration of the substance
The ratio of the intensity of the light entering the sample (Io )
to that exiting the sample (It ) at a particular wavelength is de
fined as the transmittance (T). This is often expressed as the p
ercent transmittance (%T), which is simply the transmittance
multiplied by 100. The absorbance (A) of a sample is the neg
ative logarithm of the transmittance.
% T = (Io / It ) x 100
A = - log (T)

9. 1.True deviation :
True deviations are related to the concentration of the absorbing
substance. Beers law holds good only for dilute solutions.
2. Chemical deviation :
Chemical deviations arise if the absorbing species undergo chemical
changes such as association, complex formation, dissociation,
hydrogen bonding, hydrolysis, ionization or polymerization
3. Instrumental Deviation :
Only monochromatic light gives beer’s law; use of polychromatic light
gives negative deviation.
Any fluctuations in intensity of light, change in the sensitivity of detector
, improper slit width can lead to deviation from beer lamberts law.
Instrumental deviations due to the presence of stray light

10. INSTRUMENTATION
LIGHT SOURCE
1
MONOCHROMATOR
2
SAMPLE HOLDER
3
PHOTO DETECTOR
4
READOUT DEVICE
5

12. A source must generate a beam of radiation with sufficient
power for easy detection and measurement and its output
power should be stable for reasonable periods.
A light source is required covering the range from about 200 n
m to about 800 nm.
A combination of the two is used - a deuterium (D2) lamp for
the UV part of the spectrum, and a tungsten/halogen lamp for t
he visible part.
D2 lamp produces continuous spectrum from 160 nm to the begi
nning of the visible region, 400 nm. A halogen lamp produces a
continuous spectrum of light, from near-ultraviolet to deep into t
he infrared (from 350 to 2400 nm). Instruments switch from deut
erium to tungsten at ～ 350 nm.

13. A monochromator is a device that will pass a limited
number of wavelengths of radiation.
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 int
o 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 .

14. Liquid sample is usually contained in a cell
called a cuvette.
These are small rectangular plastic, glass
or quartz containers usually sealed at one
end for holding.
They are often designed so that the light
beam travels a distance of 1 cm through
the contents.

15. The detector converts the incoming light into an electric current.
Therefore, the higher the current, the greater the intensity of the
light.
For each wavelength of light passing through the spectrometer
, the intensity of the light passing through the reference cell is
measured, Io. The intensity of the light passing through the
sample cell is also measured for that wavelength, I.
If I < Io, then obviously the sample has absorbed some of the
light. Then the photon transducer converts this into the
absorbance A of the sample.
Phototubes and photomultipliers contain a photosensitive surface
that absorbs radiation in the ultraviolet, visible, and near infrared,
producing an electric current proportional to the number of
photons reaching the transducer.
Photomultipliers and silicon photodiodes are typical detectors

16. A phototube comprises of a light-sensitive cathode and an anode
inside an evacuated quartz envelope. A photon (EM wave) entering
the tube strikes the cathode and results in ejection of an electron
which strikes the anode and results in flow of current.
The current is generally of low intensity and needs to be amplified.
The photomultiplier tube comprises of a photosensitive cathode, anode
and several dynodes. Photons entering the tube strike the cathode result
ing in emission of electrons. The resulting current often needs to be
amplified.
Photomultipliers have high sensitivity for UV and visible radiation and
have fast response times. However, they are susceptible to damage
when exposed to high intensity light. Photomultiplier tube is inherently
more sensitive than the photo tube.

17. The analog signal from the detector is displayed on an
analog meter through the position of a needle on a meter
face calibrated in percent transmission or absorbance.
Digital readouts express the signal as numbers on the face
of a meter.

18. CLASSIFICATION
A beam of radiation pass through a single cell, the reference cell is used to set the
absorbance scale at zero for the wavelength to be studied.
It is then replaced by sample cell to determine the absorbance of the sample at that
wavelength.
This was earliest design and is still use in both teaching and industrial labs.
It requires a stabilized voltage supply to avoid errors resulting from changes in the
beam intensity.

19. A double beam in space spectrophotometer utilizes two beams of light:
a reference beam and a sampling beam that passes through the sample.
Two beams are formed in space by a V-shaped mirror called beam-splitter.
This double beam spectrophotometers have two detectors that allow the two
beams to be measured at one time.

20. The beams are separated in time by a rotating sector mirror that directs the entire beam
from the monochromator first through the reference cell and then through the sample cell.
The radiation beam is alternately sent through reference and sample cells before striking
a single photo-detector. Only a matter of milliseconds separates the beams as they pass
through the two cells. Therefore, this device addresses the problem of low radiation
intensity and stability, as it alters very quickly from a reference cell to the sample cell.
The double-beam-in-time approach is generally preferred because of the difficulty in
matching the 2 detectors needed for the double-beam-in-space design.

21. Additional peaks can be observed due to
impurities in the sample and it can be
compared with that of standard raw material.
Detection of Impurities
Detecting the presence or absence of
unsaturation, the presence of hetero atoms.
Structure elucidation of organic
compounds
Quantitative analysis of compounds
that absorb UV radiation
Qualitative analysis
Absence of a band at particular wavelength
regarded as an evidence for absence of
particular group
Detect for presence or absence of
functional group
The UV radiation is passed through the reaction cell
and the absorbance changes can be observed.
Kinetics of reaction
Determination of molecular weights
of compounds
Detector for HPLC
Identification is done by comparing the
absorption spectrum with the spectra of
known compounds.

22. High sensitivity
Require only small volume
of sample
Linearity over wide range of
concentration.
Not linear for high
concentration.
Doesn’t work with compounds
that do not absorb light at
this wavelength region.
Generates significant heat and
requires external cooling
Merits
Demerits

23. It has gain a lot of popularity due to the common availability of instruments, simplicity
of use, speed, precision, accuracy of the analysis, and the relatively low cost.
In recent years, UV-Vis spectroscopy techniques have also been evaluated as useful tech
niques for monitoring several compounds simultaneously during different processes
(e.g., in line, at line).
Such methods based on UV-Vis spectroscopy will offer the possibility to provide
noninvasive and remote analysis of foods in industrial settings.
However, various barriers still hinder the growth and development of these applications
by the food industry.
Among them, the hesitancy of the food industry to accept the integration of chemistry
and mathematics and the lack of academic education and skills in the use and
application of instrumental methods based in UV-Vis spectroscopy as high-throughout
tools for process monitoring.

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Kafle, B. P. (2020). Spectrophotometry and its application in chemical analysis. Chemical Analysis and Material
Characterization by Spectrophotometry; Elsevier: New York, NY, USA, 1-16.
Kafle, B. P. (2020). Theory and instrumentation of absorption spectroscopy. Chemical Analysis and Material
Characterization by Spectrophotometry; Elsevier: New York, NY, USA, 17-38.
Nielsen, S. S. (2017). In Food Analysis. Springer, Cham.
Power, A. C., Chapman, J., Chandra, S., & Cozzolino, D. (2019). Ultraviolet-visible spectroscopy for food quality
analysis. Evaluation technologies for food quality, 91-104.
www.labmanager.com/laboratory-technology/evolution-of-uv-vis-spectrophotometers-
Evolution of UV-Vis Spectrophotometers
https://www.the-scientist.com/foundations-old/the-first-commercial-uv-vis-spectrometer-
The First Commercial UV-Vis Spectrometer