spectrofluorometer is the instrument for recording fluorescence emission and absorption spectra When a beam of light is incident on certain substances they emit visible light or radiations. This is known as fluorescence. Fluorescence starts immediately after the absorption of light and stops as soon as the incident light is cut off. The substances showing this phenomenon are known as flourescent substances.
the presentation gives knowledge about principle or fluorometry, factors that affect fluorescence including quenching instruments used in fluorometry, and the applications of fluorometry. added references in the end for more knowledge.
spectrofluorometer is the instrument for recording fluorescence emission and absorption spectra When a beam of light is incident on certain substances they emit visible light or radiations. This is known as fluorescence. Fluorescence starts immediately after the absorption of light and stops as soon as the incident light is cut off. The substances showing this phenomenon are known as flourescent substances.
the presentation gives knowledge about principle or fluorometry, factors that affect fluorescence including quenching instruments used in fluorometry, and the applications of fluorometry. added references in the end for more knowledge.
Types of crystals & Application of x raykajal pradhan
some basic information:-
A crystal lattice is a 3-D arrangement of unit cells.
Unit cell is the smallest unit of a crystal, By stacking identical unit cells, the entire lattice can be constructed
A crystal’s unit cell dimensions are defined by six numbers, the lengths of the 3 axes, a, b, and c, and the three interaxial angles, α, β and γ.
If a unit cell has the same type of atom at the corners of the unit cell but not also in the middle of the faces nor in the centre of the cell, it is called primitive and given by symbol P
7 types of crystal system details
14 bravis lattice
APPLICATION X-RAY CRYSTALLOGRAPHY
1. Structure of crystals
2. Polymer characterisation
3. State of anneal in metals
4. Particle size determination
a) Spot counting method
b) Broadening of diffraction lines
c) Low-angle scattering
5.Applications of diffraction methods to complexes
a) Determination of cis- trans isomerism
b) Determination of linkage isomerism
6.Miscellaneous applications
UV- Spectroscopy (Modern Pharmaceutical Analytical Techniques.pptxRAHUL PAL
he Principle of UV-Visible Spectroscopy is based on the absorption of ultraviolet light or visible light by chemical compounds, which results in the production of distinct spectra. Spectroscopy is based on the interaction between light and matter.
UV spectroscopy or UV–visible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum.
Introduction
working principle
fragmentation process
general rules for fragmentation
general modes of fragmentation
metastable ions
isotopic peaks
applications
Types of crystals & Application of x raykajal pradhan
some basic information:-
A crystal lattice is a 3-D arrangement of unit cells.
Unit cell is the smallest unit of a crystal, By stacking identical unit cells, the entire lattice can be constructed
A crystal’s unit cell dimensions are defined by six numbers, the lengths of the 3 axes, a, b, and c, and the three interaxial angles, α, β and γ.
If a unit cell has the same type of atom at the corners of the unit cell but not also in the middle of the faces nor in the centre of the cell, it is called primitive and given by symbol P
7 types of crystal system details
14 bravis lattice
APPLICATION X-RAY CRYSTALLOGRAPHY
1. Structure of crystals
2. Polymer characterisation
3. State of anneal in metals
4. Particle size determination
a) Spot counting method
b) Broadening of diffraction lines
c) Low-angle scattering
5.Applications of diffraction methods to complexes
a) Determination of cis- trans isomerism
b) Determination of linkage isomerism
6.Miscellaneous applications
UV- Spectroscopy (Modern Pharmaceutical Analytical Techniques.pptxRAHUL PAL
he Principle of UV-Visible Spectroscopy is based on the absorption of ultraviolet light or visible light by chemical compounds, which results in the production of distinct spectra. Spectroscopy is based on the interaction between light and matter.
UV spectroscopy or UV–visible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum.
Introduction
working principle
fragmentation process
general rules for fragmentation
general modes of fragmentation
metastable ions
isotopic peaks
applications
Uv visible spectroscopy with InstrumentationSHIVANEE VYAS
It is the branch of science that deals with the study of the interaction of matter with light.
OR
It is the branch of science that deals with the study of the interaction of electromagnetic radiation with matter.
Electromagnetic radiation is energy that is propagated through free space or through a material medium in the form of electromagnetic waves, such as radio waves, visible light, and gamma rays, etc. Electromagnetic waves consist of discrete packages of energy which are called as photons.
General Principles of Intellectual Property: Concepts of Intellectual Proper...Poonam Aher Patil
General Principles of Intellectual Property: Concepts of Intellectual
Property (IP), Intellectual Property Protection (IPP), Intellectual Property
Rights (IPR);
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
1. Mrs. Poonam Sunil Aher (M.Pharm, PhD)
Assistant Professor
Sanjivani College of Pharmaceutical Education
and Research (Autonomous),
Kopargaon, Ahmednagar-423603 (M.S.), INDIA
Mobile: +91-9689942854
UV / Visible Spectroscopy
2. Spectroscopy
It is the branch of science that deals with the study of
interaction of matter with light.
OR
It is the branch of science that deals with the study of
interaction of electromagnetic radiation with matter.
4. Electromagnetic Radiation
Electromagnetic radiation consist of
discrete packets of energy which are
called as photons.
A photon consists of an oscillating electric
field (E) & an oscillating magnetic field
(M) which are perpendicular to each
other.
5.
6. Electromagnetic Radiation
Frequency (ν):
It is defined as the number of times electrical
field radiation oscillates in one second.
The unit for frequency is Hertz (Hz).
1 Hz = 1 cycle per second
Wavelength (λ):
It is the distance between two nearest parts of
the wave in the same phase i.e. distance
between two nearest crest or troughs.
7. Electromagnetic Radiation
The relationship between wavelength &
frequency can be written as:
c = ν λ
As photon is subjected to energy, so
E = h ν = h c / λ
11. Principles of Spectroscopy
The principle is based on the
measurement of spectrum of a sample
containing atoms / molecules.
Spectrum is a graph of intensity of
absorbed or emitted radiation by sample
verses frequency (ν) or wavelength (λ).
Spectrometer is an instrument design to
measure the spectrum of a compound.
12. Principles of Spectroscopy
1. Absorption Spectroscopy:
An analytical technique which concerns
with the measurement of absorption of
electromagnetic radiation.
e.g. UV (185 - 400 nm) / Visible (400 - 800
nm) Spectroscopy, IR Spectroscopy (0.76 -
15 μm)
13. Principles of Spectroscopy
2. Emission Spectroscopy:
An analytical technique in which
emission (of a particle or radiation) is
dispersed according to some property of
the emission & the amount of dispersion
is measured.
e.g. Mass Spectroscopy
15. Interaction of EMR with matter
1.Electronic Energy Levels:
At room temperature the molecules are in the lowest
energy levels E0.
When the molecules absorb UV-visible light from EMR,
one of the outermost bond / lone pair electron is
promoted to higher energy state such as E1, E2, …En,
etc is called as electronic transition and the difference
is as:
∆E = h ν = En - E0 where (n = 1, 2, 3, … etc)
∆E = 35 to 71 kcal/mole
16. Interaction of EMR with matter
2.Vibrational Energy Levels:
These are less energy level than electronic energy levels.
The spacing between energy levels are relatively small i.e.
0.01 to 10 kcal/mole.
e.g. when IR radiation is absorbed, molecules are excited from
one vibrational level to another or it vibrates with higher
amplitude.
3. Rotational Energy Levels:
These energy levels are quantized & discrete.
The spacing between energy levels are even smaller than
vibrational energy levels.
∆Erotational < ∆Evibrational < ∆Eelectronic
18. Beer Lamberts Law:
A = ε b c
A=absorbance
ε =molar absorbtivity with units of L /mol.cm
b=path length of the sample (cuvette)
c =Concentration of the compound in solution, expressed in mol /L
20. • σ → σ* transition
1
• π → π* transition
2
• n → σ* transition
3
• n → π* transition
4
• σ → π* transition
5
• π → σ* transition
6
The possible electronic transitions are
21. • σ electron from orbital is excited to
corresponding anti-bonding orbital σ*.
• The energy required is large for this
transition.
• e.g. Methane (CH4) has C-H bond only
and can undergo σ → σ* transition and
shows absorbance maxima at 125 nm.
• σ → σ* transition
1
22. • π electron in a bonding orbital is excited
to corresponding anti-bonding orbital π*.
• Compounds containing multiple bonds
like alkenes, alkynes, carbonyl, nitriles,
aromatic compounds, etc undergo π →
π* transitions.
e.g. Alkenes generally absorb in the
region 170 to 205 nm.
• π → π* transition
2
23. • Saturated compounds containing atoms
with lone pair of electrons like O, N, S
and halogens are capable of n → σ*
transition.
• These transitions usually requires less
energy than σ → σ* transitions.
• The number of organic functional groups
with n → σ* peaks in UV region is small
• n → σ* transition
3
24. • An electron from non-bonding orbital is
promoted to anti-bonding π* orbital.
• Compounds containing double bond
involving hetero atoms (C=O, C≡N, N=O)
undergo such transitions.
• n → π* transitions require minimum
energy and show absorption at longer
wavelength around 300 nm.
• n → π* transition
4
25. •These electronic transitions are forbidden
transitions & are only theoretically
possible.
•Thus, n → π* & π → π* electronic
transitions show absorption in region
above 200 nm which is accessible to UV-
visible spectrophotometer.
•The UV spectrum is of only a few broad of
• σ → π* transition
5
• π → σ* transition 6
&
28. Chromophore
The part of a molecule responsible for imparting color, are called as
chromospheres.
OR
The functional groups containing multiple bonds capable of absorbing
radiations above 200 nm due to n → π* & π → π* transitions.
e.g. NO2, N=O, C=O, C=N, C≡N, C=C, C=S, etc
29. Auxochrome
The functional groups attached to a chromophore which modifies the ability of
the chromophore to absorb light , altering the wavelength or intensity of
absorption.
OR
The functional group with non-bonding electrons that does not absorb
radiation in near UV region but when attached to a chromophore alters the
wavelength & intensity of absorption.
33. • When absorption maxima (λmax) of a
compound shifts to longer wavelength, it
is known as bathochromic shift or red
shift.
• The effect is due to presence of an
auxochrome or by the change of solvent.
• e.g. An auxochrome group like –OH, -OCH3
causes absorption of compound at longer
wavelength.
• Bathochromic Shift (Red Shift)
1
34. • In alkaline medium, p-nitrophenol shows
red shift. Because negatively charged
oxygen delocalizes more effectively than
the unshared pair of electron.
p-nitrophenol
λmax = 255 nm λmax = 265 nm
• Bathochromic Shift (Red Shift)
1
OH
N
+ O
-
O
OH
-
Alkaline
medium
O
-
N
+ O
-
O
35. • When absorption maxima (λmax) of a
compound shifts to shorter wavelength, it
is known as hypsochromic shift or blue
shift.
• The effect is due to presence of an group
causes removal of conjugation or by the
change of solvent.
• Hypsochromic Shift (Blue Shift)
2
36. • Aniline shows blue shift in acidic medium,
it loses conjugation.
Aniline
λmax = 280 nm λmax = 265 nm
• Hypsochromic Shift (Blue Shift)
2
NH2
H
+
Acidic
medium
NH3
+
Cl
-
37. • When absorption intensity (ε) of a
compound is increased, it is known as
hyperchromic shift.
• If auxochrome introduces to the
compound, the intensity of absorption
increases.
Pyridine 2methylpyridine
λmax = 257 nm λmax = 260 nm
• Hyperchromic Effect
3
N N CH3
38. • When absorption intensity (ε) of a
compound is decreased, it is known as
hypochromic shift.
Naphthalene 2-methyl naphthalene
ε = 19000 ε = 10250
CH3
• Hypochromic Effect
4
39. Wavelength ( λ )
Absorbance
(
A
)
Shifts and Effects
Hyperchromic shift
Hypochromic shift
Red
shift
Blue
shift
λmax
41. Principle
The UV radiation region extends from 10 nm to 400 nm and the visible
radiation region extends from 400 nm to 800 nm.
Near UV Region: 200 nm to 400 nm
Far UV Region: below 200 nm
Far UV spectroscopy is studied under vacuum condition.
The common solvent used for preparing sample to be analyzed is either
ethyl alcohol or hexane.
44. Five Basic Optical Instrument Components
1) Source – A stable source of radiant energy at the desired wavelength (or
range).
2) Wavelength Selector – A device that isolates a restricted region of the
EM spectrum used for measurement (monochromators, prisms & filters).
3) Sample Container – A transparent container used to hold the sample
(cells, cuvettes, etc).
4) Detector/Photoelectric Transducer – Converts the radiant energy into a
useable signal (usually electrical).
5) Signal Processor & Readout – Amplifies or attenuates the transduced
signal and sends it to a readout device as a meter, digital readout, chart
recorder, computer, etc.
47. LIGHT SOURCES
Various UV radiation sources are as follows
a. Deuterium lamp
b. Hydrogen lamp
c. Tungsten lamp
d. Xenon discharge lamp
e. Mercury arc lamp
Various Visible radiation sources are as follow
a. Tungsten lamp
b. Mercury vapour lamp
c. Carbonone lamp
48. Wavelength Selectors
Wavelength selectors output a limited, narrow, continuous group of
wavelengths called a band.
Two types of wavelength selectors:
A) Filters
B) Monochromators
A)Filters –
Two types of filters:
a) Interference Filters
b) Absorption Filters
49. Cont..
B. Monochromators
Wavelength selector that can continuously scan a broad range of
wavelengths.
Used in most scanning spectrometers including UV, visible, and IR
instruments.
Refractive type
PRISM TYPE
Reflective type
Diffraction type
GRATING TYPE
Transmission Type
50. SAMPLE COMPARTMENT
Spectroscopy requires all materials in the beam path other than the analyte should
be as transparent to the radiation as possible.
The geometries of all components in the system should be such as to maximize the
signal and minimize the scattered light.
The material from which a sample cuvette is fabricated controls the optical window
that can be used.
Some typical materials are:
Optical Glass - 335 - 2500 nm
Special Optical Glass – 320 - 2500 nm
Quartz (Infrared) – 220 - 3800 nm
Quartz (Far-UV) – 170 - 2700 nm
51. Detectors
After the light has passed through the sample, we want to be able to detect and
measure the resulting light.
These types of detectors come in the form of transducers that are able to take
energy from light and convert it into an electrical signal that can be recorded, and if
necessary, amplified.
Three common types of detectors are used
Barrier layer cells
Photo emissive cell detector
Photomultiplier
52. SUMMARY
Types of source, sample holder and detector for various EM region
REGION SOURCE SAMPLE
HOLDER
DETECTOR
Ultraviolet Deuterium lamp Quartz/Fused
silica
Phototube, PM
tube, diode
array
Visible Tungsten lamp Glass/Quartz Phototube, PM
tube, diode
array
53. Types of dosage form
Two types
Single component dosage form
Multi component dosage form
54. Method for single component dosage form:
1. Calibration method or beer lamberts law method 2.Method of least
Square
3. Single point standardization method
4. Double point standardization method
55. Beer law method or Calibration curve method:
1. prepare standard stock
2. prepare dilution of standard stock
3. calculate range
4. calculate wavelength
5. Perform the assay
6. Draw the graph
7. Calculate concentration by using regression line equation Y = m x + c
8. calculate the unknown concentration by graph
9. Calculate the purity of drug by using Beer law A=abc
56. This method is modified in new form
Method of least squares:
57.
58. 4. Double point standardization method:
C TEST= ( A test- A STD1)( C STD 1- C STD2) + CSTD1(
ASTD1-ASTD2)
______________________________________
(ASTD1- ASTD2)
60. (a) Simultaneous equation method:
If a sample contains two absorbing drugs (X and Y)
each of which absorbs at the λ-max of the other (λ1
and λ2), it may be possible to determine both the drugs
by the simultaneous equations method.
61. The information required is
The absorptivities of X at λ1 and λ2, aX1 and aX2.
The absorptivities of Y at λ1 and λ2, aY1 and aY2.
The absorbances of the diluted sample at λ1 and λ2, A1
and A2.
Let, Cx and Cy be the concentration of X and Y
respectively in the sample.
The absorbance of the mixture is the sum of the
individual absorbances of X and Y
62. At λ1 A1 = aX1* Cx + aY1* Cy …………..(1)
At λ2 A2 = aX2* Cx + aY2* Cy …………..(2)
Multiply the equation (1) with aX2 and (2) with aX1
A1 aX2 = aX1 Cx aX2 + aY1 Cy aX2 …………(3)
A2 aX1 = aX2 Cx aX1+ aY2 Cy aX1 ………….(4)
A1 aX2 - A2 aX1 = aY1 Cy aX2 - aY2 Cy aX1
A1 aX2 - A2 aX1 = Cy (aY1 aX2 - aY2 aX1)
Cy = (A1 aX2 - A2 aX1) / (aY1 aX2 - aY2 aX1) ……….(5)
Same way we can derive
Cx = (A2 aY1 – A1 aY2) / (aY1 aX2 - aY2 aX1)………... (6)
These equations are known as simultaneous equations and by solving
these simultaneous equations we can determine the concentration
of X and Y in the sample.
63. (b) Absorbance ratio method:
The absorbance ratio method is a
modification of the simultaneous equations procedure.
In the quantitative assay of two components in
admixture by the absorbance ratio method,
absorbances are measured at two wavelengths, one
being the λ-max of one of the components (λ2) and
other being a wavelength of equal absorptivity of two
components (λ1), i.e. an iso-absorptive point.
64. At λ1 A1 = aX1* Cx + aY1* Cy …………… (1)
At λ2 A2 = aX2* Cx + aY2* Cy…………....(2)
Now divide (2) with (1)
A2/A1 = (aX2* Cx + aY2* Cy)/(aX1* Cx + aY1* Cy)
Divide each term with (Cx + Cy)
A2/A1 = (aX2* Cx + aY2* Cy) / (Cx + Cy) (aX1* Cx + aY1* Cy) / (Cx + Cy)
Put Fx = Cx / (Cx + Cy) and Fy = Cy / (Cx + Cy)
A2/A1 = [aX2 Fx + aY2 Fy] / [aX1 Fx + aY1Fy]
Where Fx is the fraction of X and Fy is the fraction of Y i.e. Fy = 1-Fx
Therefore,
A2/A1 = [aX2 Fx + aY2 (1-Fx)] / [aX1 Fx + aY1(1-Fx)]
= [aX2 Fx + aY2 – aY2Fx] / [aX1 Fx + aY1 – aY1Fx]
65. At iso-absorptive point
aX1 = aY1 and Cx = Cy
There fore A2/A1 = [aX2 Fx + aY2 – aY2Fx] / aX1
= (aX2 Fx/ aX1) + (aY2/ aX1) –( aY2Fx/ aX1)
Let Qx = aX2/aX1 , Qy = aY2/aY1 and absorption ratio Qm = A2/A1
Qm = Fx (Qx-Qy) + Qy
Fx = (Qm – Qy) / (Qx – Qy) ………………………..(3)
From the equations (1) A1 = aX1 (Cx + Cy)
there fore Cx + Cy = A1 / aX1
There fore Cx = (A1/aX1) – Cy ……………………(4)
From the equation (3)
Cx / (Cx + Cy) = (Qm – Qy) / (Qx – Qy)
There fore Cx / (A1 / aX1) = (Qm – Qy) / (Qx – Qy)
There fore Cx = [(Qm – Qy) / (Qx – Qy)] X (A1 / aX1) …………(5)
66. (c) Geometric correction method:
A number of mathematical correction procedures have been
developed which reduce or eliminate the background irrelevant
absorption that may be present in samples of biological origin.
The simplest of this procedure is the three point geometric
procedure, which may be applied if the irrelevant absorption is
linear at the three wavelengths selected.
67. If the wavelengths λ 1, λ 2 and λ 3 are selected to that
the background absorbances B 1 , B 2 and B 3 are
linear, then the corrected absorbance D of the drug
may be calculated from the three absorbances A 1 , A 2
and A 3 of the sample solution at λ 1, λ 2 and λ 3
respectively as follows,
Let v D and w D be the absorbance of the drug alone in
the sample solution at λ 1 and λ 3 respectively, i.e. v
and w are the absorbance ratios vD/D and wD/D
respectively.
B 1 = A 1 – vD, B 2 = A 2 –D and B 3 = A 3 –wD
68. Let y and z be the wavelengths intervals (λ 2 – λ 1 ) and (λ 3 - λ 2 )
respectively
D= y(A 2 -A 3 ) + z(A 2 – A 1 ) / y (1-w) + z(1-v)
This is a general equation which may be applied in any situation where A
1, A 2 and A 3 of the sample, the wavelength intervals y and z and the
absorbance ratio v and w are known.
69. (d) Orthogonal polynomial method:
The technique of orthogonal polynomials is another mathematical
correction procedure, which involves more complex calculations than the
three-point correction procedure. The basis of the method is that an
absorption spectrum may be represented in terms of orthogonal functions
as follows
A(λ ) = p P (λ ) + p1 P1 (λ ) + p2 P2 (λ ) ….. pn Pn (λ )
Where A denotes the absorbance at wavelength λ belonging to a set
of n+1 equally spaced wavelengths at which the orthogonal
polynomials, P (λ ) , P1 (λ ), P2 (λ ) ….. Pn (λ ) are each defined.
70. (e)Derivative Spectroscopy:
For the purpose of spectral analysis in order to relate
chemical structure to electronic transitions, and for
analytical situations in which mixture contribute
interfering absorption, a method of manipulating the
spectral data is called derivative spectroscopy.
Derivative spectrophotometry involves the conversions
of a normal spectrum to its first, second or higher
derivative spectrum. In the context of derivative
spectrophotometry, the normal absorption spectrum is
referred to as the fundamental, zero order, or D 0
spectrum.
71. INVENTION
This technique was first described by
Hammond and Price in 1953, followed by the
work of Morrison and French et al.
Theoretical aspects have been discussed by
several authors and a number of reviews
concerning these aspects and the performance
of the technique have been published.
72. A first-order derivative is
the rate of change of
absorbance with respect to
wavelength. A firstorder
derivative starts and
finishes at zero.
It also passes through zero
at the some wavelength as
max of the absorbance
band.
Either side of this point are
positive and negative bands
with maximum and
minimum at the some
wavelengths as the
inflection points in the
absorbance band.
This bipolar function is
characteristic of all odd-
order derivatives.
73.
74. The first derivative D 1 spectrum is a plot of the rate of change of
absorbance with wavelength against wavelength i.e. a plot of the
slope of the fundamental spectrum against wavelength or a plot of
dA/dλ vs. λ. . The maximum positive and maximum negative slope
respectively in the D spectrum correspond with a maximum and a
minimum respectively in the D 1 spectrum. The λmax in D
spectrum is a wavelength of zero slope and gives dA/dλ = 0 in the
D 1 spectrum.
The second derivative D 2 spectrum is a plot of the curvature of
the D spectrum against wavelength or a plot of d 2 A/ dλ 2 vs. λ.
The maximum negative curvature in the D spectrum gives a
minimum in the D 2 spectrum, and the maximum positive
curvature in the D spectrum gives two small maxima called
satellite bands in the D 2 spectrum. The wavelength of maximum
slope and zero curvature in the D spectrum correspond with cross-
over points in the D 2 spectrum.
75. OBTAINING DERIVATIVE SPECTRA
• Derivative spectra can be obtained by optical, electronic, or
mathematical methods.
• Optical and electronic techniques were used on early UV-
Visible spectrophotometers but have largely been superseded
by mathematical techniques.
• The advantages of the mathematical techniques are that
derivative spectra may be easily calculated and recalculated
with different parameters, and smoothing techniques may be
used to improve the signal-to-noise ratio.
76. OPTICALAND ELECTRONIC TECHNIQUES
The main optical technique is wavelength modulation,where
the wavelength of incident light is rapidly modulated over
a narrow wavelength range by an electromechanical
device. The first and second derivatives may be generated
using this technique
The electronic method suffers from the disadvantage that the
amplitude and wavelength shift of the derivatives varies
with scan speed, slit width, and resistance-capacitance
gain factor.
77. MATHEMATICAL TECHNIQUES
To use mathematical techniques the spectrum is first
digitized with a sampling interval of wavelenth. The size
depends on the natural bandwidth (NBW) of the bands
being processed and bandwidth of the instrument used to
generate the data. Typically, for UV-Visible spectra, the
NBW is in the range 10 to 50 nm.
Firstderivative spectra may be calculated simply by taking
the difference in absorbance between two closely
spaced wavelengths for all wavelengths
78. Where the derivative amplitude, D, is calculated for a
wavelength intermediate between the two absorbance
wavelengths.
For the second-derivative determination three closely-spaced
wavelength values are used
Savitzky and Golay developed a very efficient method to
perform the calculations and this is the basis of the
derivatization algorithm in most commercial instruments.
Other techniques for calculating derivatives, for example,
using Fourier Transforms, are available but not
commercially popular.
79. QUANTIFICATION If we assume that the zero-
order spectrum obeys Beer’s
law, there is a similar linear
relationship between
concentration and amplitude
for all orders of derivative
For single component
quantification the selection of
wavelengths for derivative
spectra is not as simple as for
absorbance spectra because
there are both positive and
negative peaks.
For the even order derivatives
there is a peak maximum or
minimum at the same
wavelength as the absorbance
spectrum but for the odd-
order derivatives this
wavelength is a zero crossing
point.
80. Spectral Discrimination as a qualitative fingerprinting
technique to accentuate small structural differences between
nearly identical spectra
Spectral resolution enhancement as a technique for increasing
the apparent resolution of overlapping spectral bands in order
to more easily determine the number of bands and their
wavelengths
Quantitative Analysis as a technique for the correction for
irrelevant background absorption and as a way to facilitate
multicomponent analysis.
USES
81. ADVANTAGES
•An effective enhancement of resolution, which can be useful
to separate two or more components with overlapping spectra.
• A discrimination in favour of the sharpest features of a
spectrum, used to eliminate interferences by broad band
constituents.
82. (f)Difference Spectroscopy:
Difference spectroscopy provides a sensitive method for detecting
small changes in the environment of a chromophore or it can be
used to demonstrate ionization of a chromophore leading to
identification and quantitation of various components in a mixture.
The essential feature of a difference
spectrophotometric assay is that the measured value is the
difference absorbance (Δ A) between two equimolar solutions of
the analyte in different forms which exhibit different spectral
characteristics.
The criteria for applying difference spectrophotometry to the
assay of a substance in the presence of other absorbing substances
are that:
A)Reproducible changes may be induced in the spectrum of the
analyte by the addition of one or more reagents.
B) The absorbance of the interfering substances is not altered by the
reagents.
83. The simplest and most commonly employed technique for altering the
spectral properties of the analyte properties of the analyte is the
adjustment of the pH by means of aqueous solutions of acid, alkali or
buffers
A B
A)The Spectrum of compound in A(acid) and B(Base)
B) The difference spectrum of B relative to A
84.
85.
86.
87. Application of UV spectroscopy:
Qualitative & Quantitative Analysis:
It is used for characterizing aromatic compounds and conjugated olefins.
It can be used to find out molar concentration of the solute under study.
Detection of impurities:
It is one of the important method to detect impurities in organic solvents.
Eg. In cyclohexane, benzene is impurity and it is checked at 255 nm
Eg detection of impurity in starting material of nylon
Detection of isomers are possible.
88. Quantitative analysis:
UV absorption spectroscopy can be used for the quantitative
determination of compounds that absorb UV radiation. This determination
is based on Beer’s law which is as follows.
A = log I0 / It = log 1/ T = – log T = abc = εbc
Where ε is extinction co-efficient, c is concentration, and b is the length
of the cell that is used in UV spectrophotometer.
Other methods for quantitative analysis are as follows.
a. calibration curve method
b. simultaneous multicomponent method
c. difference spectrophotometric method
d. derivative spectrophotometric method
89. Qualitative analysis
UV absorption spectroscopy can characterize those types of compounds
which absorbs UV radiation. Identification is done by comparing the
absorption spectrum with the spectra of known compounds.
UV absorption spectroscopy is generally used for characterizing aromatic
compounds and aromatic olefins.
Quantitative analysis of pharmaceutical substances
Many drugs are either in the form of raw material or in the form of
formulation. They can be assayed by making a suitable solution of the
drug in a solvent and measuring the absorbance at specific wavelength.
Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at the
wavelength 284 nm.
90. Chemical kinetics:
To study the chemical reaction and kinetic study of reactant and product
Charge transfer transition reaction:
To study the charge transfer transition reaction of product by using total
wave function
Tautomeric equilibrium:
to study the tautomeric equilibrium of keto and enol forms.
Eg: ethyl acetoacetate in this keto forms show absorption band at 275 nm
Structure of charcoal:
To study the structure of charcoal
91. Molecular weight determination
Molecular weights of compounds can be measured spectrophotometrically
by preparing the suitable derivatives of these compounds.
For example, if we want to determine the molecular weight of amine then
it is converted in to amine picrate. Then known concentration of amine
picrate is dissolved in a litre of solution and its optical density is measured
at λmax 380 nm. After this the concentration of the solution in gm moles
per litre can be calculated by using the following formula.
"c" can be calculated using above equation, the weight "w" of amine
picrate is known. From "c" and "w", molecular weight of amine picrate can
be calculated. And the molecular weight of picrate can be calculated
using the molecular weight of amine picrate.
92. Dissociation constants of acids and bases.
PH = PKa + log [A-] / [HA]
From the above equation, the PKa value can be
calculated if the ratio of [A-] / [HA] is known at a
particular PH. and the ratio of [A-] / [HA] can be
determined spectrophotometrically from the graph
plotted between absorbance and wavelength at
different PH values.
93. Reference Books
Introduction to Spectroscopy
Donald A. Pavia
Elementary Organic Spectroscopy
Y. R. Sharma
Practical Pharmaceutical Chemistry
A.H. Beckett, J.B. Stenlake
99. LIGHT SOURCES
Various UV radiation sources are as follows
a. Deuterium lamp
b. Hydrogen lamp
c. Xenon discharge lamp
Various Visible radiation sources are as follow
a. Tungsten filament lamp
b. Mercury arc lamp
c. Carbonone lamp
100.
101. For ultra violet region
1 Hydrogen discharge lamp
Consist of two electrode contain in Hydrogen filled silica glass or quartz envelop
Gives continuous spectrum in region 185-380nm. above 380nm emission is not
continuous
102. Working:
In HDL the a continuous spectrum is produced by electrical
excitation of hydrogen gas at low pressure( 0.2-0.5 torr)
In this continuous spectrum first initial formation of excited
hydrogen species by ionization of hydrogen.
H2(gas) ionization H2* dissociation H2’ + H2’’
103. Deuterium lamps:
Deuterium arc lamps measure in the UV region
190 - 370 nm
As Deuterium lamps operate at high
temperatures, normal glass housings cannot be
used for the casing. Instead, a fused quartz, UV
glass, or magnesium fluoride envelope is used.
When run continuously typical lamp life for a
Deuterium lamp is approximately 1000 hours
Deuterium lamps are always used with a
Tungsten halogen lamp to allow measurements
to be performed in both the UV and visible
regions.
Working is same like hydrogen lamp. Deuterium
gas is used.
105. 3. xenon discharge lamp:
It is also called xenon arc lamp
In this lamp xenon gas is filled under the pressure 20-30 atm
This gas is passed through 2 electrodes which are separated
by 8 mm distance
The intense arc is formed between two electrodes by
supplying high voltage
This lamp give spectrum in the 200-1000 nm therefore it can
work in uv and visible region
106.
107. Tungsten filament lamp:
This lamp is used in visible region
It gives continuous spectrum in 350-2000 nm region
It is also work in IR region
It can not used in UV region
The lamp consist of tungsten filament which act as
electrode
This electrode is evacuated in quartz envelop
The tungsten gas is filled in the envelop
The filament is heated upto 3000k and creates plasma or
light in the visible region
108.
109. Mercury arc lamp:
In this lamp inert mercury gas is filled in the form of vapor
This lamp is used in UV and Visible region
Excitation of mercury atoms done by high electrical voltage
and it gives high intensity of continuous lines
110. Filters &Monochromator
The Monochromator/Filter will select a narrow portion of the spectrum (the band
pass) of a given source
FILTERS ARE OF TWO TYPES:
Absorption Filters
Interference Filters
MONOCHROMATORS ARE OF TWO TYPES:
Refractive type
PRISM TYPE
Reflective type
Diffraction type
GRATING TYPE
Transmission Type
111. FILTERS
1.ABSORPTION FILTERS(Gelatin filter)
Absorption filters, commonly manufactured from dyed glass or pigmented gelatine
resins is and sandwiched between two glass plate
Band widths are extremely large {30 – 250 nm}
Combining two absorbance filters of different λmax
112. The most common type of gelatine filter is constructed by sandwiching a thin layer
of dyed gelatine of the desired colour between two thin glass plates.
114. INTERFERENCE FILTERS:
These are used to select wavelengths more
accurately by providing a narrow band
pass typically of around 10nm
These filters rely on optical interference
(destructive wave addition) to provide
narrow bands of radiation.
115.
116. Interference filter consists of a dielectric spacer film made
up of CaF2, MgF2 between two parallel reflecting films.
As light passes from one medium to the other the direction
and wavelength of light can be changed based on the index
of refraction of both mediums involved and the angle of the
incident and exiting light
Due to this behaviour, constructive and destructive
interference can be controlled by varying the thickness (d)
of a transparent dielectric material between two semi-
reflective sheets
As light hits the first semi-reflective sheet, a portion is
reflected, while the rest travels through the dielectric to be
bent and reflected by the second semi-reflective sheet
120. Material used in prism monochromator:
1. Glass – visible region
2. Quartz: uv and visible
3. Borosilicate glass: both region
4.Sodium chloride: IR
5.Potassium bromide: IR
121. Three types of prism monochromator:
1. Refractive type monochromator: Cornu
Prism
2. Reflective Type monochromator: Littrow
prism
3. Dispersive power type monochromator:
Bunsen Prism
122. 1. Cornu prism:
It is also called refractive prism
It consist of 30˚ two prism
So the total optical angle of prism is 60˚
Here two right handed quartz prism 30˚ and left handed 30˚
quartz prism are fabricated together
In this prism incident light strike on the prism gives minimum
deviation but it gives maximum dispersion
Here refracted light is directly focused on exit slit
123. 2. Littrow Prism :
It is also called reflective type prism
It is 30˚ optical prism where one of coated by reflective film
like aluminum, copper or gold material
The reflective light is back to pass through the prism and
immerse on the same direction of the light source
The incident light is can not cross the second surface of
prism because the surface is coated with reflective material
125. 3. Bunsen prism:
It is simple 60 prism
It is dispersive type prism
126. GRATING MONOCHROMATOR
Gratings are rulings made on glass, Quartz or alkyl halides
Depending upon the instrument no. of rulings per mm defers
If it is UV-Visible no. of gratings per mm are more than 3600.
127. Types of grating monochromator
Two types
1. Diffraction or reflective type grating
2. transmission type grating
130. The mechanism is that diffraction produces reinforcement.
The rays which are incident on the grating gets reinforced with the reflected rays
and hence resulting radiation has wavelength governed by equation
m λ =b(sin I + sin r)
m – order
λ – desired wavelength
b – grating spacing
i – angle of incidence
r - angle of diffraction
131. Eg: Czerny turner grating monochromator:
It is reflective type monochromator
132. TRANSMISSION TYPE GRATING:
It is similar to diffraction grating, but refraction produces instead of reflection
Refraction produces reinforcement
The wavelength of radiation produced by transmission grating can be expressed by
d sin Φ
λ = d = 1/lines per cm
m
133. Eg: Litrrow Blazed grating monochromator:
It is also called Claised Monochromator.
The configuration of monochromator in a such way that the
entrance slits are positioned at the 90 degree angle to
focusing and collimated lens. And the entrance slit and exit
slit are in the same direction
The diffraction angle is calculated by following formula
d( sin α +sin β )= n λ
α is angle of incident light
β is angle of diffracted light
λ is wavelength
134.
135. SAMPLE COMPARTMENT
Spectroscopy requires all materials in the beam path other than the
analyte should be as transparent to the radiation as possible.
The geometries of all components in the system should be such as
to maximize the signal and minimize the scattered light.
The material from which a sample cuvette is fabricated controls the
optical window that can be used.
Some typical materials are:
Optical Glass - 335 - 2500 nm
Special Optical Glass – 320 - 2500 nm
Quartz (Infrared) – 220 - 3800 nm
Quartz (Far-UV) – 170 - 2700 nm •
136. Ideal characteristics of sample holder:
1. minimizes reflection
2. path length 1 cm
3 calibrated in sixe
4 should be 0.1 to 10 cm long
5 use square holders mostly in spectrometric
experiment
6 use cylindrical holders in colorimetric experiment
137. Precaution while handling to holders:
1 don’t touch holder surface
Always clean surface by soft tissue paper
before use
Always calibrate the cuvette before use
Don’t dry the cuvette in hot air oven or direct
on flame
When corrosive solution used in experiment
then clean the cuvette by using methanol and
dip the cuvette in methanol for 15 min
138. DETECTORS
After the light has passed through the sample, we want to be able to detect and
measure the resulting light.
These types of detectors come in the form of transducers that are able to take
energy from light and convert it into an electrical signal that can be recorded, and if
necessary, amplified.
Three common types of detectors are used
Barrier layer cells
Photo emissive cell detector
Photomultiplier
139. BARRIER LAYER CELL or PHOTO VOLTAIC CELL
It constitutes
The barrier layer cell consist of semiconductor material such as Selenium, which is
deposited on a strong metal base of iron.
Thin layer of silver or gold is sputtered over the surface of semiconductor which act as
a second electron collector
metallic base plate like iron or aluminium which acts as one electrode( cathode) and
silver layer act as anode
This total assembly is fitted in glass or plastic window
140.
141. Principle:
When the radiation is incident upon the surface of selenium, electrons are generated
at the selenium- silver surface and the electrons are accumulated on silver and they
collected by the silver semiconductor material .
This accumulation at the silver surface creates an electric voltage difference is
between the silver surface and the base of the cell.
A photocurrent will flow which is directly proportional to the intensity of incident
radiation beam.
This detector does not require external power supply.
It is directly connected to galvanometer.
142. Advantage
1. very robust in construction
2. it is cheap
3. rugged
4. no require external power supply
5 good for portable instrument
6 It shows linear relationship
Disadvantages:
1. It is not very sensitive to reading
2. It shows fatigue
143. Photo emissive cells Detector
/Photocell
Phototubes are also known as photo emissive cells.
A phototube consists of an evacuated glass bulb or tube
It contains two electrodes
There is light sensitive cathode inside it.
The inner surface of cathode is coated with light sensitive layer such as potassium
oxide and silver oxide or selenium.
When radiation is incident upon a cathode, photoelectrons are emitted.
These are collected by an metal wire anode.
The current which is created between anode and cathode is measured as radiation
falling on the detector.
Then these are returned via external circuit. And by this process current is amplified
and recorded.
144.
145. Advantages:
It is sensitive
The signal is easily amplified
Disadvantages:
It shows some dark current (means it shows no signal
after some time)
146. The photomultiplier tube
The photomultiplier tube is a most commonly used detector in
UV spectroscopy.
A photomultiplier tube is an evacuated glass tube which
contains one photocathode and 9-16 electrodes known as
dynodes.
The surface each dynode is Be-Cu, Cs-Sb.
A photon of radiation entering the tube strikes the cathode,
causing the emission of several electrons.
147. Principle:
When radiation falls on metal surface of the photocathode, it emits electrons.
These electrons are accelerated towards the first dynode which is kept as 90V at
positive voltage. Which is 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.
Eventually all electrons are finally collected at the anode.
148. By this time, each original photon has produced 106 - 107 electrons.
The final current is amplified or recorded in the terms of signal and response
Photomultipliers are very sensitive to UV and visible radiation.
They have fast response times.
Intense light damages photomultipliers;
they are limited to measuring low power radiation
The transit time between absorption of photon and arrival of the shower of electrons
is typically 10-100 sec.
149.
150.
151. Advantages:
It is extreme sensitive than other detector
The emission of photon in less time so this process require 10-100 sec
Each photon produce 106-10 7 electron
Disadvantages:
Due to intense light it damages the PMT
It require high voltage power supply
Instrument may damage if excessive current drawn from final anode
This detector can not response to low DC current
153. Spectrophotometric titration. Definition: The process
of determining the quantity of a sample by adding
measured increments of a titrant until the end-point, at
which essentially all of the sample has reacted, is
reached.
Principle is a method to measure how much a chemical
substance absorbs light by measuring the intensity of
light as a beam of light passes through sample solution.
The basic principle is that each compound absorbs or
transmits light over a certain range of wavelength.
154. one species in a complexation titration absorbs electromagnetic radiation,
we can identify the end point by monitoring the titrand’s absorbance at a
carefully selected wavelength.
For example, we can identify the end point for a titration of Cu2+ with
EDTA, in the presence of NH3 by monitoring the titrand’s absorbance at a
wavelength of 745 nm, where the Cu(NH3)4
2+ complex absorbs strongly.
At the beginning of the titration the absorbance is at a maximum. As we
add EDTA the concentration of Cu(NH3)4
2+, and thus the
absorbance, decreases as EDTA displaces NH3. After the equivalence point
the absorbance remains essentially unchanged.
155. The resulting spectrophotometric titration is shown
below in panel (a).
Note that the titration curve’s y-axis is not the actual
absorbance, A, but a corrected absorbance, Acorr
Acorr = A × (VEDTA + VCu)/VCu
where VEDTA and VCVCuu are, respectively, the volumes
of EDTA and Cu.
Correcting the absorbance for the titrand’s dilution
ensures that the spectrophotometric titration curve
consists of linear segments that we can extrapolate to
find the end point.
156.
157. Examples of spectrophotometric titration curves:
(a) only the titrand absorbs;
(b) only the titrant absorbs;
(c) only the product of the titration reaction absorbs;
(d) both the titrand and the titrant absorb;
(e) both the titration reaction’s product and the titrant absorb;
(f) only the indicator absorbs. The red arrows indicate the end points for
each titration curve.