Spectroscopy and it's applications as well as it's types like Infrared spectroscopy and ultraviolet spectroscopy and principle of spectroscopy why we use spectroscopy.
This presentation gives you thorough knowledge about the IR Spectroscopy. This include basic principle, type of vibrations, factors influencing vibrational frequency, instrumentation and applications of IR Spectroscopy. This is the most widely used technique for identifying unknown functional group depending on the vibrational frequency.
Ultraviolet-visible (UV-Vis) spectrophotometry is a technique used to measure light absorbance across the ultraviolet and visible ranges of the electromagnetic spectrum. When incident light strikes matter it can either be absorbed, reflected, or transmitted. The absorbance of radiation in the UV-Vis range causes atomic excitation, which refers to the transition of molecules from a low-energy ground state to an excited state.
This presentation gives you thorough knowledge about the IR Spectroscopy. This include basic principle, type of vibrations, factors influencing vibrational frequency, instrumentation and applications of IR Spectroscopy. This is the most widely used technique for identifying unknown functional group depending on the vibrational frequency.
Ultraviolet-visible (UV-Vis) spectrophotometry is a technique used to measure light absorbance across the ultraviolet and visible ranges of the electromagnetic spectrum. When incident light strikes matter it can either be absorbed, reflected, or transmitted. The absorbance of radiation in the UV-Vis range causes atomic excitation, which refers to the transition of molecules from a low-energy ground state to an excited state.
Sepration of molecules on the basis of applied Electric Field
Categorized into 1) Zone Electrophoresis 2) Moving Boundary Electrophoresis
We can seprate macromolecules (DNA , RNA, PROTEINS )on the basis of their charge, size shape & molecular weight
SPECTROSCOPY is defined as the study of the interactions between radiations and matter as function of wavelength λ .
Interactions with particle radiation or a response of a material to an altering field
or varying frequency.
SPECTRUM : A plot of the response as a function of wavelength or more commonly frequency is referred to as spectrum.
SPECTROMETRY : It is measurement of these responses and an instrument which performs such measurements is a spectrophotometer or spectrograph, although
these terms are more limited in use to original field of optics from which the
concept sprang.
Gas chromatography-mass spectrometry (GC-MS) is the synergistic combination of two analytical method to separate and identify different substances within a test sample.
Gas chromatography separates the components of a mixture in time.
Mass spectrometer provides information that aids in the identification and structural elucidation of each component.
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
IR SPECTROSCOPY, INTRODUCTION, PRINCIPLE, THEORY, FATE OF ABSORBED RADIATION, FERMI RESONANCE, FINGERPRINT REGION, VIBRATIONS, FACTORS AFFECTING ABSORPTION OF IR RADIATION, SAMPLING TECHNIQUES, APPLICATIONS OF IR SPECTROSCOPY.
Electrophoresis:
Electrophoresis is separation technique based on movement of charge particle in an electric field.
Movement of charge particles can be determined by following formula--
V= Eq/f
Where,
V= Velocity of the charged particle;
E= electric field of the molecule;
q= Net charge of the molecule; and
f= Frictional co-efficient of the molecule
Types of electrophoresis:
1. Agarose gel electrophoresis ;
2. Poly-acryl amide gel electrophoresis [PAGE];
3. Sodium do-decyl sulphate Poly- acrylamide gel electrophoresis [SDS-PAGE] ;
4. Two dimensional –Poly-acrylamide gel electrophoresis [2D-PAGE];
5. Pulse field gel electrophoresis [PFGE];
6. Capillary gel electrophoresis [CGE]; and
7. Disc electrophoresis for Protein.
Application of electrophoresis:
1. Estimation of the DNA molecule.[ Agarose , PAGE ]
2. Analysis of PCR product. [ Agarose ]
3. Separation of restricted genomic DNA and RNA. [Agarose and PAGE respectively]
4. Conformation of newly isolated DNA .[Agarose]
5. Separation of most small fragments of DNA. [PAGE]
6. In forensic science.[Agarose , PAGE, SDS-PAGE, 2D PAGE ,Capillary gel electrophoresis , PFGE]
8. In determining molecular wt. of protein.[SDS-PAGE].etc
It would be use full to All Needy People. It involve information about NMR Spectroscopy ( a spectroscopic techniques), factors influencing , proton NMR and their applications of NMR as well as Nuclear magnetic imaging.
Sepration of molecules on the basis of applied Electric Field
Categorized into 1) Zone Electrophoresis 2) Moving Boundary Electrophoresis
We can seprate macromolecules (DNA , RNA, PROTEINS )on the basis of their charge, size shape & molecular weight
SPECTROSCOPY is defined as the study of the interactions between radiations and matter as function of wavelength λ .
Interactions with particle radiation or a response of a material to an altering field
or varying frequency.
SPECTRUM : A plot of the response as a function of wavelength or more commonly frequency is referred to as spectrum.
SPECTROMETRY : It is measurement of these responses and an instrument which performs such measurements is a spectrophotometer or spectrograph, although
these terms are more limited in use to original field of optics from which the
concept sprang.
Gas chromatography-mass spectrometry (GC-MS) is the synergistic combination of two analytical method to separate and identify different substances within a test sample.
Gas chromatography separates the components of a mixture in time.
Mass spectrometer provides information that aids in the identification and structural elucidation of each component.
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
IR SPECTROSCOPY, INTRODUCTION, PRINCIPLE, THEORY, FATE OF ABSORBED RADIATION, FERMI RESONANCE, FINGERPRINT REGION, VIBRATIONS, FACTORS AFFECTING ABSORPTION OF IR RADIATION, SAMPLING TECHNIQUES, APPLICATIONS OF IR SPECTROSCOPY.
Electrophoresis:
Electrophoresis is separation technique based on movement of charge particle in an electric field.
Movement of charge particles can be determined by following formula--
V= Eq/f
Where,
V= Velocity of the charged particle;
E= electric field of the molecule;
q= Net charge of the molecule; and
f= Frictional co-efficient of the molecule
Types of electrophoresis:
1. Agarose gel electrophoresis ;
2. Poly-acryl amide gel electrophoresis [PAGE];
3. Sodium do-decyl sulphate Poly- acrylamide gel electrophoresis [SDS-PAGE] ;
4. Two dimensional –Poly-acrylamide gel electrophoresis [2D-PAGE];
5. Pulse field gel electrophoresis [PFGE];
6. Capillary gel electrophoresis [CGE]; and
7. Disc electrophoresis for Protein.
Application of electrophoresis:
1. Estimation of the DNA molecule.[ Agarose , PAGE ]
2. Analysis of PCR product. [ Agarose ]
3. Separation of restricted genomic DNA and RNA. [Agarose and PAGE respectively]
4. Conformation of newly isolated DNA .[Agarose]
5. Separation of most small fragments of DNA. [PAGE]
6. In forensic science.[Agarose , PAGE, SDS-PAGE, 2D PAGE ,Capillary gel electrophoresis , PFGE]
8. In determining molecular wt. of protein.[SDS-PAGE].etc
It would be use full to All Needy People. It involve information about NMR Spectroscopy ( a spectroscopic techniques), factors influencing , proton NMR and their applications of NMR as well as Nuclear magnetic imaging.
Introduction,Instrumentation, Classification of electronic transitions, Substituent and solvent effects, Classification of electronic transitions
Substituent and solvent effects
Applications of UV Spectroscopy
UV spectral study of alkenes
UV spectral study of poylenes
UV spectral study of α, β-unsaturated carbonyl
UV spectral study of Aromatic compounds
Empirical rules for calculating λmax.
Applications of UV Spectroscopy, Empirical rules for calculating λmax.
UV-Visible spectroscopy is considered as an important tool in the analytical chemistry.
Most powerful tool available for the study of atomic and molecular structure.
- Most commonly used techniques in clinical as well as chemical laboratories.
- Used for the qualitative analysis and identification of chemicals.
ain use is for quantitative determination of different organic and inorganic compounds in solution.
Basically, spectroscopy is related to the interaction of light with matter.
As light is absorbed by matter, the result is an increase in the energy content of the atoms or molecules.
The absorption of visible or ultraviolet light by a chemical compound will produce a distinct spectrum.
UV-Visible light range- 200-800 nm
Visible range: 400-800 nm
UV range: 200-400 nm
principle, application and instrumentation of UV- visible Spectrophotometer Ayetenew Abita Desa
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Prepare a presentation or a paper using research, basic comparative analysis, data organization and application of economic information. You will make an informed assessment of an economic climate outside of the United States to accomplish an entertainment industry objective.
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The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
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2. Spectroscopy
• Spectroscopy is the study of the interaction
between matter and electromagnetic radiation.
• Historically, spectroscopy originated through the study
of visible light dispersed according to its wavelength
by a prism
• The electromagnetic spectrum is the range of frequencies of
electromagnetic radiation and their respective wavelengths and photon
energies
3. Principle of spectroscopy
• The term "spectroscopy" defines a large number of
techniques that use radiation to obtain information on the
structure and properties of matter.
• The basic principle shared by all spectroscopic techniques is
to shine a beam of electromagnetic radiation onto a sample,
and observe how it responds to such a stimulus.
4.
5.
6. • The history of spectroscopy began with Isaac Newton's optics
experiments (1666–1672).
• Newton applied the word "spectrum" to describe the rainbow of colors
that combine to form white light and that are revealed when the white
light is passed through a prism.
• During the early 1800s, Joseph von Fraunhofer made experimental
advances with dispersive spectrometers that enabled spectroscopy to
become a more precise and quantitative scientific technique.
8. • Applications
• Spectroscopy is used as a tool for studying the structures
of atoms and molecules. The large number of wavelengths
emitted by these systems makes it possible to investigate their
structures in detail.
9. • Spectroscopy also provides a precise analytical method for
finding the constituents in material having unknown
chemical composition.
• In a typical spectroscopic analysis, a concentration of a few
parts per million of a trace element in a material can be detected
through its emission spectrum
10.
11. How to classify spectroscopy
• Spectroscopy can be defined by the type of radiative energy involved.
The intensity and frequency of the radiation allow for a measurable
spectrum.
• Electromagnetic radiation is a common radiation type and was the
first used in spectroscopic studies.
• Both infrared (IR) and near IR use electromagnetic radiation, as well
asmicrowave techniques.
12. • Another way of classifying spectroscopy is by the nature of
the interaction between the energy and the material. These
interactions include absorption, emission.
13.
14. IR Spectroscopy
• Range of electromagnetic spectrum that is used is Infrared
radiation.
• Infrared is makeup of different radiations.
• The measurement of the interaction of infrared radiation with
matter by absorption, reflection.
• Used to find functional group in molecules can liquid
gaseous forms.
15.
16.
17. • After absorbing energy molecules vibrate. Vibration of two typ
• Streching
1. Symetrical
2. Assymetrical
• Bending
1. Scissoring
2. Wagging
3. Rocking
4. Twisting
18.
19. • Bonds show different vibrations at different
wavelengths.
• Different functional groups absorbs different
wavelength of light so show different peaks.
20. • Formula to find vibrations in linear molecules.
21.
22. Spectrum
• Spectrum have two main regions.
• Absorbtion region:-
• Individual peaks we can identify easily.
• Fingerprinting region:-
• Multiple peaks
• We can’t identify easily
• But by matching with spectrum library.
• Represents bands of bending and stretching.
23. Applications
• To establish Identity of two compounds.
• To determine the structure of new compound from its functional
group.
• To determine nature of contaiminants in a sample.
• Some advanced physical properties of material.
24. • Vibrational energy depends upon following:-
• Mass of atom
• Strength of bonds and bond distance
• The arrangement of atom within molecule.
25. UV visible spectroscopy
• Why we need UV visible spectroscopy.
• We need to find concentration of different substances compounds
mixtures.
• It can also tell us about chemical groups but data is not reliable.
• Also use in kinetic study in enzymatic activity.
• Also known as colour emmitery.
• Determining molar concenteration
• Determining ppm
26.
27. • Detector can be
• Photodiode
• PMT
• Photomultiplier
• Monochromator consists of
• 2 slits
• 1 prism
• First slit fall light to prism
• Second slit allow one wavelength of light to pass.
28. • Cuvette with organic compounds
or proteins.
• Proteins absorb wavelength if
260 NM.
• More protein more absorbtion.
• Less intensity of reflected light.
• I decrease.
• Transmitance ~ 1/absorbance
• Transmitance depends upon
concentration of molecule.
29. • Detector only detect
transmitance than how we
find absorbance.
• So we get graph.
30. • To convert a value from percent transmittance (%T)
to absorbance, use the following equation:
• Absorbance = 2 – log(%T)
• Example: convert 56%T to absorbance:
• 2 – log(56) = 0.252 absorbance units.
• Absorbance=10-1(concenteration× path length of cuvette)
• Concenteration less =transmitance more
• A=€ ×C×l
31. • If we know absorbance than can
calculate concenteration.
• C=A/€×l
Because absorbance and
concenteration does not have
linear relationship.
In order to know unknown sample
we need a linear graph.
32. NMR
• Nuclear magnetic resonance
• We determine chemical and physical properties of different organic
and inorganic molecules.
• Let say protein different atoms arrange in different ways.
• If we take example of hydrogen it consists of 1 proton and 1 electron.
• Proton is surrounded by electron.
• Proton spin it can behave as magnet.
• It is not just for Hydrogen also for other elements.
33. • So this proton behave as magnet
and it repel and try to rotate
other atom present near it.
• NMR machine creates magnetic
field.
• It directs this magnet in
whatever direction it is to stable
state.
34.
35. • How it is going to help us in determination of of structures.
• It tells us about special arrangement of atom with respect to each
other.
• Cl -C-H2
• HCl-C-H
• These are two different structures but IR can’t differencite between
these two.
• But pattern of graph obtain help us to understand arrangements.
36. •Cl H-C-H CH3
•As Hydrogen arrange in electromagnetic field of NMR
less energy is required to change its direction because
it is shielded. But chlorine also present there it
attracts all atoms of carbon towards it so more energy
for Hydrogen alpha.
•For hydrogen beta less energy because chlorine
electronegative effect is less.
37. • Emission spectroscopy
• It uses to find how much concenteration of element present in
sample.
• Or which element is present in sample sample we can use food
products like noodles or cold drinks or something else.
• Atomic absorbtion
• Use to detect metallic elements that are present.
• Detect calcium magnesium potassium in serum.
• Lead in petrol.
38.
39. • Nabolizer:-
• Convert solid sample to aerosols.
• Flame role:-
• Dissolution :-evaporate the solvent
• Vapourization:-solute convert to gas
• Atomization:-dissociation produce atoms
• Excitation of atoms.
• Emission of atoms.
• Emission release different wavelength of light so unique spectrum
obtained.
40.
41. 1-Fluorescenece spectroscopy
• Fluorescence spectroscopy uses higher energy photons to excite a
sample,which will then emit lower energy photos. This techniques
has become popular for its biochemical and medical applications.
• Fluorescence spectroscopy is used in, among others,
biochemical, medical, and chemical research fields for
analyzing organic compounds.
• There has also been a report of its use in differentiating
malignant skin tumors from benign.
42. • In the field of water research, fluorescence spectroscopy can
be used to monitor water quality by detecting organic
pollutants.
• Recent advances in computer science and machine learning
have even enabled detection of bacterial contaminaton of
water.
43. 2- X-ray Spectroscopy:
• X-ray of sufficient frequencies interact with material and excite the
atoms contained. Excitation radiations are absorbed or evolved if vice
versa occurs. X-ray absorption and emission spectroscopy is used in
chemistry and material sciences to determine elemental competition
and chemical bonding.
• Very good and versatile techniques but a little complex. Overall X-ray
diffraction techniques is one that is used most widely for bond length
and angle measurements.
44.
45. 7- Raman spectroscopy:
• Raman spectroscopy; is a spectroscopic technique typically used to determine
vibrational modes of molecules, although rotational and other low-frequency
modes of systems may also be observed.
• Raman spectroscopy is commonly used in chemistry to provide a structural
fingerprint by which molecules can be identified.
46.
47.
48.
49.
50. Spectrometer
• Spectrometer is apparatus to measure spectrum show intensity as
function of
• Wavelength
• Frequency
• Energy
• Mass
52. Optical spectrometer
•Show intensity of light as function of wavelength
and frequency.
•The deflection is produced either by refraction in
a prism or diffraction in a diffraction grating.
53. Mass spectrometer
• It is analytic instrument is analytical instruments that is used
to:-
• Identify the amount and type of chemicals present in sample
by measuring the mass to charge ratio abundance of gas
phase ions.
54.
55. Time of light spectrometer
• Determining the time of flight between two detectors
• If volocity is known masses can be determined.