Ultraviolet-visible spectroscopy involves promoting electrons from the highest occupied molecular orbital to the lowest unoccupied molecular orbital when molecules absorb electromagnetic radiation. This causes different types of electronic transitions that can be observed based on wavelength shifts. The technique follows Beer's law where absorbance is directly proportional to concentration and path length. It is used to determine conjugation and identify functional groups in molecules.
UV -Vis Spectrophotometry- Principle, Theory, Instrumentation and Application...Dr. Amsavel A
UV -Vis Spectrophotometry- Principle, Theory, Instrumentation and Application in Pharmaceutical Industry Dr. A. Amsavel.
UV &Visible Spectroscopy-Absorption Theory
Electronic Transitions
Beer- Lambert Law
Chromophores & Auxochrome
Factors Influence the Absorption
UV-Vis Spectrophotometer-Instrumentation
Operation of the Spectrophotometer
Qualification & Calibration
Application
spectrophotometry, ultra violet absorption, infra red atomic absorption.priya tamang
A spectrophotometer is a photometer that can measure the intensity of light as a function of its wavelength. Single beam and double beam are the two major classes of spectrophotometers. Linear range of absorption and spectral bandwidth measurement are the important features of spectrophotometers.
In Single Beam Spectrophotometers, all the light passes through the sample. To measure the intensity of the incident light the sample must be removed so that all the light can pass through. This type of spectrometer is usually less expensive and less complicated. The single beam instruments are optically simpler and more compact, znc can also have a larger dynamic range.
In a Double Beam Spectrophotometer, before it reaches the sample, the light source is split into two separate beams. One beam passes through the sample and the second one is used for reference. This gives an advantage because the reference reading and sample reading can take place at the same time.
In transmission measurements, the spectrophotometer quantitatively compares the amount of light passing through the reference and test sample. For reflectance, it compares the amount of light reflecting from the test and reference sample solutions.
Many spectrophotometers must be calibrated before they start to analyse the sample and the procedure for calibrating spectrophotometer is known as "zeroing." Calibration is done by using the reference substance, and the absorbencies of all other substances are measured relative to the reference substance. % transmissivity (the amount of light transmitted through the substance relative to the initial substance) is displayed on the spectrophotometer.
http://www.redicals.com
The spectrophotometer technique is to measures light intensity as a function of wavelength.
• Measures the light that passes through a liquid sample
• Spectrophotometer gives readings in Percent Transmittance (%T) and in Absorbance (A)
UV -Vis Spectrophotometry- Principle, Theory, Instrumentation and Application...Dr. Amsavel A
UV -Vis Spectrophotometry- Principle, Theory, Instrumentation and Application in Pharmaceutical Industry Dr. A. Amsavel.
UV &Visible Spectroscopy-Absorption Theory
Electronic Transitions
Beer- Lambert Law
Chromophores & Auxochrome
Factors Influence the Absorption
UV-Vis Spectrophotometer-Instrumentation
Operation of the Spectrophotometer
Qualification & Calibration
Application
spectrophotometry, ultra violet absorption, infra red atomic absorption.priya tamang
A spectrophotometer is a photometer that can measure the intensity of light as a function of its wavelength. Single beam and double beam are the two major classes of spectrophotometers. Linear range of absorption and spectral bandwidth measurement are the important features of spectrophotometers.
In Single Beam Spectrophotometers, all the light passes through the sample. To measure the intensity of the incident light the sample must be removed so that all the light can pass through. This type of spectrometer is usually less expensive and less complicated. The single beam instruments are optically simpler and more compact, znc can also have a larger dynamic range.
In a Double Beam Spectrophotometer, before it reaches the sample, the light source is split into two separate beams. One beam passes through the sample and the second one is used for reference. This gives an advantage because the reference reading and sample reading can take place at the same time.
In transmission measurements, the spectrophotometer quantitatively compares the amount of light passing through the reference and test sample. For reflectance, it compares the amount of light reflecting from the test and reference sample solutions.
Many spectrophotometers must be calibrated before they start to analyse the sample and the procedure for calibrating spectrophotometer is known as "zeroing." Calibration is done by using the reference substance, and the absorbencies of all other substances are measured relative to the reference substance. % transmissivity (the amount of light transmitted through the substance relative to the initial substance) is displayed on the spectrophotometer.
http://www.redicals.com
The spectrophotometer technique is to measures light intensity as a function of wavelength.
• Measures the light that passes through a liquid sample
• Spectrophotometer gives readings in Percent Transmittance (%T) and in Absorbance (A)
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The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
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In this webinar you will learn how your organization can access TechSoup's wide variety of product discount and donation programs. From hardware to software, we'll give you a tour of the tools available to help your nonprofit with productivity, collaboration, financial management, donor tracking, security, and more.
2. Ultraviolet and visible spectroscopy also known as
electronic spectroscopy is used to measure the
number of conjugation of double bond and aromatic
conjugation within the molecule.
It involves the promotion of electrons from
HOMO to LUMO (HOMO means Highest
Occupied Molecular Orbital whereas LUMO
means Lowest Unoccupied Molecular Orbital).
The HOMO-LUMO gap decrease as the
conjugation increase.
3. The ultraviolet region corresponds to 400-200 nm and visible region to 800-
400 nm.
UV-visible spectroscopy based on Beer-Lambert law.
Where,
log
Io⁄I = ε. 𝑙. 𝑐 or ϵ = 𝐴/𝑐 𝑙
Io = intensity of incident light
I = intensity of transmitted light
ε = molar absorptivity
l = path length of sample
c = concentration of sample
A = Absorbance
Absorbance (A): It is reciprocal of Transmittance
1
Absobance (A) = Optical density (D) = log
T
or log
Io
I
4. When molecule is getting excited by the absorption of electromagnetic
radiation in UV-visible region then its electrons are promoted from
ground state to excited state or from bonding orbital to anti-bonding
orbital.
Types of electronic transitions
𝜎 − 𝜎* Transition: transition of an electron from bonding sigma orbital (𝜎)
to anti-bonding sigma orbital ( *), is represented by 𝜎 − 𝜎 * transition. For
example, alkanes because in alkane all the atoms are held together with
sigma bond.
𝑛 − 𝜎* & 𝑛 − 𝜋* Transition: Transitions from non-bonding molecular (𝑛)
orbital to anti-bonding sigma orbital or anti-bonding pi orbital (𝜋*), are
represented by 𝑛 − 𝜎 * or 𝑛 − 𝜋 * transition respectively. These transition
required less energy than 𝜎 − 𝜎 * transition. For example, alkyl halide,
aldehydes, ketones etc.
𝜋 − 𝜋 * Transition: This type of transition generally show in unsaturated
molecules like alkenes, alkynes, aromatics, carbonyl compounds etc. This
transition required less energy as compare to 𝑛 − 𝜎* transition
5.
6. Bathochromic shift: It is also known as Red
shift, in this case absorption shift towards
longer wavelength (𝜆max).
Hypsochromic shift: It is also known as Blue
shift, in this case absorption shift towards
shorter wavelength (𝜆max).
Hyperchromic shift: Intensity of absorption
maximum (𝜀max) increases.
Hypochromic shift: Intensity of absorption
maximum (𝜀max) decrease.
7.
8. Chromophores: Chromophore is a covalently
unsaturated group absorbed electromagnetic
radiation in UV-visible region and impart color
to the compound. For example, C=C, C≡C,
benzene, NO2 etc.
Auxochromes: Auxochrome is a saturated
group (containing lone pair of electron), when
auxochrome attach to the Chromophore
absorption shift towards longer wavelength.
For example, -OH, -SH, -NH etc
9.
10. Conjugation
Absorption shift towards longer wavelength, if
double bonds (chromophore) present in the
molecule are in conjugation. For example
In compound ‘A’, double bonds are in
conjugation therefore ‘A’ possessing higher
wavelength (𝜆max) as compare to compound ‘B’
(non-conjugated derivative).
11.
12. Effect of solvent:
In polar solvent, 𝜋 − 𝜋* transitions shift
towards longer wavelength (Red shift),
because dipole interactions with polar
solvent molecules lower the energy of
excited state (𝜋*) than that of the ground
state (𝜋* orbital’s are stabilized by hydrogen
bonding with polar solvent).
13.
14. Double bond extending conjugation 30
R alkyl substituent or ring residue 5
Exocyclic double bond 5
Polar groupings:
-OCOCH3 0
-OR 6
-Cl, -Br 5
-NR2 60
19. Spectrophotometer is a kind of spectrometer, which
measures the transmittance or absorbance of a sample
as a function of wavelength, when light of certain
intensity and frequency range is passed through the
sample. Unlike a spectrometer (which is any
instrument that can measure the properties of light
over a range of wavelengths), a spectrophotometer
measures only the intensity of light as a function of its
wavelength. The key components of a
spectrophotometer are:
Light source
Monochromator
Sample area
Detector and Recorder
20. The most suitable sources of light are
Deuterium lamp which emits the light in the UV-
region (160-375 nm)
Tungsten lamp or tungsten-halogen lamp which
emits radiation in the Visible and near infrared
regions (350-2500 nm)
Xenon arc lamp which emits radiation in the range
190-800 nm
Light emitting diodes (LED) which emits radiation
in the visible range 400-800 nm.
The instruments automatically swap lamps when
scanning between the UV and UV-VIS regions.
21. The main function of the monochromator is to
disperse the beam of light obtained from the
primary source, into its component (Figure 3).
The principle components of monochromator
are
An entrance slit
A collimating lens
A dispersing device
A focusing lens
An exit slit
22. The radiation emitted from the primary source
(polychromatic radiation) enters the
monochromator through the entrance slit. The
beam is collimated and then strikes the
dispersing element (Prism or grit) at a
particular angle. Two types of dispersion
devices viz. prisms and holographic gratings
(Figure 4) are commonly used in UV-visible
spectrophotometers.
23.
24.
25. One of the two divided beams is passed
through the sample solution and the other
beam is passed through the reference solution.
Although, the samples for recording spectra
are most commonly liquids, but the absorbance
of gases and even of solids can be measured.
Both sample and
reference solutions are placed in a transparent
cell, known as cuvette. The cuvettes are
rectangular in shape, and usually have an
internal width of 1 cm
26.
27. A detector converts a light signal into an electrical signal. After the
beams are passed through the sample under study as well as the
reference cell, the intensities of the respective transmitted beams
are then compared over the whole wavelength range of the
instrument. Generally two photocells are used as detector in UV
spectrometer to record the spectra. One of the photocell receives
the beam from sample cell and second detector receives the beam
from the reference. The intensity of the radiation from the
reference cell is stronger than the beam of sample cell. This results
in the generation of pulsating or alternating currents in the
photocells.
Spectrophotometers consist of either a photomultiplier tube
detector or a photodiode detector.
The commonly used detector in UV-Vis spectroscopy is
photomultiplier tube (Figure 6). It is m consists of three
components:
28. In it’s functioning, when a photon of radiation
strikes the cathode, emission of several electrons
take place. These emitted electre then accelerated
towards the many dynodes. The first dynode is
90V more positive than the cathode. The electrons
strike the first dynode, causing the emission of
several electrons for each incident electron. These
electrons are then accelerated towards the second
dynode, to produce more electrons which are
accelerated towards dynode three and so on.
Finally all the electrons are collected at the anode.
Several dynodes are arranged
29.
30. When the radiation passes through a solution, some amount of light is absorbed. Suppose I0 is
theintensity of the incident radiation and I, the intensity of the transmitted radiation.
The amount of radiation absorbed can be measured
byTransmittance T = I/I0
% Transmittance = T×100
Absorbance A = Log10 I0/I= Log10
1/T
If the entire light passes through a solution without any absorption, then absorbance is
zero.In this case, the percent transmittance is 100%.
If all the light is absorbed, then absorption is infinite and the percent transmittance is zero %.
Beer-Lambert Law: It gives a linear relationship between absorbance and concentration
of an absorbing species. This law states that the absorption is directly proportional to the
concentration of absorbing substance and the length of the path of radiation through the
sample. It is independent of the intensity of the incident light and each successive unit layer
absorbs an equal fraction of light incident on it.
A = ɛcb
Log10 I0/I=
ɛcb
Where A is the sample’s Absorbance value at specific wavelength or frequency
ε is the molar absorptivity or the molar extinction coefficient of the
substanceb is the path length of the sample in cm
c is the concentration of the solute in mol L-1
.
The value of ε absorptivity coefficient is constant for a particular material at a particular
wavelength.
31. Under certain conditions Beer-Lambert law fails to
maintain a linear relationship between absorbance
and concentration of the solution.
Deviations in absorptivity coefficients at high
concentrations (>0.01M) due to electrostatic
interactions between molecules in close proximity.
Scattering of light due to particles present in the
sample.
Chemical deviations due to the specific chemical
species of the sample under investigation.
Non-monochromatic radiation
Fluorescence or phosphorescence of the sample