Mass spectrometry is a technique that determines molecular mass and elemental composition of compounds. It works by ionizing molecules using a beam of electrons, which causes the molecules to fragment into ions of various m/e ratios. These ions are then separated by their mass-to-charge ratios using electric or magnetic fields. Common applications include determining molecular structures of organic molecules and identifying unknown compounds.
MASS SPECTROSCOPY ( Molecular ion, Base peak, Isotopic abundance, Metastable ...Sachin Kale
CONTENT:
Molecular Ion Peak
Significance of Molecular ion & Graphically Method
Base Peak
Isotopic Abundance
Metastable Ion
Significance of Metastable ion
Nitrogen Rule & graphs
Formulation of Rule
MASS SPECTROSCOPY ( Molecular ion, Base peak, Isotopic abundance, Metastable ...Sachin Kale
CONTENT:
Molecular Ion Peak
Significance of Molecular ion & Graphically Method
Base Peak
Isotopic Abundance
Metastable Ion
Significance of Metastable ion
Nitrogen Rule & graphs
Formulation of Rule
MS Fragmentation Process and Application of MS.pdfDr. Dinesh Mehta
Fragmentation process:
Bombardment of molecules by an electron beam with energy between 10-15ev usually results in the ionization of molecules by removal of one electron (Molecular ion formation).
Ion exclusion chromatography is a technique,introduced by Wheaton and Bauman, used to separate ionic compounds from non-ionic compounds and to separate mixtures of acids.
MS Fragmentation Process and Application of MS.pdfDr. Dinesh Mehta
Fragmentation process:
Bombardment of molecules by an electron beam with energy between 10-15ev usually results in the ionization of molecules by removal of one electron (Molecular ion formation).
Ion exclusion chromatography is a technique,introduced by Wheaton and Bauman, used to separate ionic compounds from non-ionic compounds and to separate mixtures of acids.
This presentation contains a simple discussion about the basic principles, Instrumentation, Various ionization techniques, mass analyzers, Mass detectors, Fragmentation, and various peak observed in Mass spectra(Molecular ion peak, Metastable peak, Base peak etc)
And application of Mass spectroscopy on various field.
Introduction, Basic Principles, Terminology, Instrumentation, Ionization techniques (EI, CI, FAB, MALDI, and ESI), Mass Analyzer (Magnetic sector instruments, Quadrupole, TOF, and ICR ), and Applications of Mass Spectrometry.
Uploaded By: Mr. Shubham sutradhar (masters in
pharmaceutical Chemistry).
Mass spectroscopy & it's instrumentations, Ionization Techniques, Mass Spectroscopic Analyzers & it's applications. above topics are discussed in a brief format.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
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.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
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.
Richard's entangled aventures in wonderlandRichard 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.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
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.
8. Spectroscopy
Basic Principle
Mass spectroscopy is the most accurate method for
determining the molecular mass of the compound and its
elemental composition.
In this technique, molecules are bombarded with a beam of
energetic electrons.
The molecules are ionised and broken up into many fragments,
some of which are positive ions.
Each kind of ion has a particular ratio of mass to charge, i.e.
m/e ratio(value). For most ions, the charge is one and thus, m/e
ratio is simply the molecular mass of the ion.
8
10. Spectroscopy
Ionisation
The atom is ionised by knocking one or more electrons
off to give a positive ion. (Mass spectrometers always
work with positive ions).
The particles in the sample (atoms or molecules) are
bombarded with a stream of electrons to knock one or more
electrons out of the sample particles to make positive ions.
10
11. Spectroscopy
Most of the positive ions formed will carry a charge
of +1.
These positive ions are persuaded out into the rest of the
machine by the ion repeller which is another metal plate
carrying a slight positive charge.
1
1
13. Spectroscopy
The positive ions are repelled away from the positive
ionisation chamber and pass through three slits with voltage
in the decreasing order.
The middle slit carries some intermediate voltage and the
final at ‘0’ volts.
All the ions are accelerated into a finely focused beam.
1
3
14. Spectroscopy
Deflection
The ions are then deflected by a magnetic field
according to their masses. The lighter they are, the
more they are deflected.
The amount of deflection also depends on the
number of positive charges on the ion -The more the
ion is charged, the more it gets deflected.
14
15. Spectroscopy
Different ions are deflected by the magnetic field by
different amounts. The amount of deflection depends
on:
The mass of the ion: Lighter ions are deflected
more than heavier ones.
The charge on the ion: Ions with 2 (or more)
positive charges are deflected more than ones with only
1 positive charge.
1
5
16. Spectroscopy
Detection
The beam of ions passing through the machine is detected
electrically.
When an ion hits the metal box, its charge is neutralised
by an electron jumping from the metal on to the ion.
16
Only ion stream B makes it right through the machine to
the ion detector.
The other ions collide with the walls where they will
pick up electrons and be neutralised.
They get removed from the mass spectrometer by the
vacuum pump.
17. Spectroscopy
That leaves a space amongst the electrons in the
metal, and the electrons in the wire shuffle along to fill
it.
A flow of electrons in the wire is detected as an
electric current which can be amplified and recorded.
The more ions arriving, the greater the current.
1
7
25. Spectroscopy
The Sample Inlet System
Batch Inlets
The batch inlet system is considered the most
common and simplest inlet system. Normally, the
inside of the system is lined with glass to elude losses
of polar analyte by adsorption.
This system externally volatizes the sample which
leaks into an empty ionization region. Boiling points up
to 500 degrees C of gaseous and liquid samples can
be used on typical systems.
The system's vacuum contains a sample pressure of
10-4 to 10-5 Torr. Liquids are introduced using a
microliter syringe into a reservoir; gases are enclosed
in a metering area that is confined between two valves
before being expanded into a reservoir container.
26. Spectroscopy
Liquids that have boiling points lower than 500
degrees C can not be used in the system because
the reservoir and tubing need to be kept at high
temperatures by ovens and heating tapes. This is
to ensure that the liquid samples are transformed
to the gaseous phase and then leaked through a
metal or glass diaphragm containing pinholes to
the ionization area.
27. Spectroscopy
The Direct Probe Inlet:
A direct probe inlet is for small quantities of
sample, solids, and nonvolatile liquids. Solids and
nonvolatile liquids are injected through a probe, or
sample holder.
The probe is inserted through a vacuum lock.
Unlike the batch inlet, the sample will need to be
cooled and/or heated on the probe.
The probe is placed extremely close (a few
millimeters) to the ionization source, where the slit
leads to the spectrometer,
28. Spectroscopy
Electrophoretic Inlets
Chromatographic systems and Capillary
Electrophoretic units are often coupled with mass
spectrometers in order to allow separation and
identification of the components in the sample. If
these systems and units are linked with a mass
spectrometer, then other specialized inlets,
Electrokinetic and
Pressure injection, are required.
Electrokinetic and pressure injection controls the
amount of volume injected by the duration of the
injection, which typically range between 5 to 50 nL.
29. Spectroscopy
ION SOURCE
Since the mass analyzer utilizes only gaseous ions i.e.,
starting point of mass spectrometric analysis is
formation of gaseous analyte ions.
• Non –Volatile solids are first converted in to gases and
from the gaseous sample the ions are produced in a Box
like enclosure called Ion Source.
Function
Produces ion without mass discrimination
sample.
Accelerates ions into the mass analyzer.
of the
30. Spectroscopy
Desorption
A phenomenon whereby a substance is released from or
through a surface.
Sorption
A process whereby one substance attached to another. It
can be of two types
1 Adsorption
Adhesion of atoms ions or molecules from a gas liquid
or dissolved solid to a surface. This process create a
adsorbate on the surface of adsorbent.
2 Absorption
A process in which atoms ions or molecules are taken up
by a bulk phase i.e solid liquid or gas. Molecules are
taken up by the volume not by the surface.
31. Spectroscopy
Catogories of Ion sources
Gas Phase Sources
Electron Impact Ionization (EI)
Chemical Ionization (CI)
Field Ionizations (FI)
Desorption Sources
Field Desorption (FD)
Electrospray Ionization (ESI)
Matrix assisted desorption/Ionisation (MALDI)
Plasma desorption (PD)
Fast Atom Bombardment (FAB)
Thermospray Ionization (TS)
Secondary Ion Mass Spectrometry (SIMS)
32. Spectroscopy
• Electron impact (EI) ionization
Electron impact (EI) is the classical ionization method
in mass spectrometry.
• It is the most widely used and highly developed
method.
• It is also known as Electron bombardment or
Electron Ionization.
33. Spectroscopy
CONSTRUCTION & WORKING:
Electron impact ionization source consists of a ionizing chamber
which is maintained at a pressure of 0.005 torr and temperature of
200 ± 0.25 degrees.
Electron gun is located perpendicular to chamber.
Electrons are emitted from a glowing filament (tungsten or
rhenium) and accelerated by a potential of 70 eV applied between
the filament and anode.
These electrons are drawn in the ionization chamber through
positively charged slits.
• The number of electrons is controlled by filament temperature
and energy of energy is controlled by filament potential.
The sample is brought to a temperature high enough to produce
molecular vapors.
• The gaseous Neutral molecules then pass through the molecular
leaks and enter the ionization
34. Spectroscopy
The gaseous sample and the electrons collide at right
angles in the chamber and ions are formed by exchange
of energy during these collisions between electron
beam and sample molecules
Since the ionization energy of most of the organic
molecules is 15eV an electron is expelled to produce a
radical cation.
At hard ionization event i.e at 70 e V molecule ions are
fragmented.
The positive ions formed in the chamber are drawn out
by a small potential difference (usually 5eV) between
the large repeller plate (positively charged) and first
accelerating plate (negatively charged).
35. Spectroscopy
ADVANTAGES
Gives molecular mass and also the fragmentation
pattern of the sample.
Extensive fragmentation and consequent large number
of peaks gives structural information.
Gives reproducible mass spectra.
DISADVANTAGES
Sample must be thermally stable and volatile.
A small amount of sample is ionized (1 in 1000
molecules).
Unstable molecular ion fragments are formed so readily
that are absent from mass spectrum.
39. Spectroscopy
Schematic representation of an electron
ionization ion source.
sample pressure in the ion source
is about 10-5 torr
about every 1/1000
molecule is ionized
only cations
the sample is heated up
until a sufficient vapour
pressure is obtained
40. Spectroscopy
Chemical ionization
In chemical ionization, the ionization of the analyte is
achieved by interaction of it’s molecules with ions of
a reagent gas in the chamber or source.
Chemical ionization is carried out in an instrument
similar to electron impact ion source with some
modifications such as:-
Addition of a vacuum pump.
Narrowing of exit slit to mass analyzer to maintain
reagent gas pressure of about 1 torr in the ionization
chamber.
Providing a gas inlet.
41. Spectroscopy
It is a two part process.
• In the first step
A reagent gas is ionized by Electron Impact ionization in
the source.
The primary ions of reagent gas react with additional
gas to produce stabilized reagent ions.
In the second step, the reagent ions interact with
sample molecules to form molecular ions.
• In this technique the sample is diluted with a large
excess of reagent.
Gases commonly used as reagent are low molecular
weight compounds such as Methane, tertiary Isobutane,
Ammonia, Nitrous oxide, oxygen and hydrogen etc.
42. Spectroscopy
TYPES OF CI:
Depending upon the type of
categorized as:-
1. Positive Chemical Ionization
2. Negative Chemical Ionization
1. Positive Chemical Ionization
ions formed CI is
In this technique positive ions of the sample are
produced.
In positive chemical ionization, gases such as Methane,
Ammonia, Isobutane etc are used.
43. Spectroscopy
For example,
Ammonia is used as reagent gas.
First ammonia radical cations are generated by electron
impact and this react with neutral ammonia to form
ammonium cation (reactive species of ammonia CI).
NH4+reacts with the sample molecules by proton transfer
to produce sample ions
44. Spectroscopy
Negative Chemical Ionization
Negative chemical ionization is counterpart of Positive
chemical ionization.
In this technique, negative ions of the sample are
formed.
Oxygen and Hydrogen are used as reagent gasses.
This method is used for ionization of highly
electronegative samples.
45. Spectroscopy
ADVANTAGES
Used for high molecular weight compounds.
Used for samples which undergo rapid fragmentation
in EI.
LIMITATIONS
Not suitable for thermally unstable and non-volatile
samples.
Relative less sensitive then EI ionization.
Samples must be diluted with large excess of reagent gas
to prevent primary interaction between the electrons and
sample molecules.
47. Spectroscopy
Field Ionization
FI is used to produce ions from volatile compounds
that do not give molecular ions by EI.
It produces molecular ions with little or no
fragmentation.
Application of very strong electric field induces
emission of electrons.
FI utilizes 10-micron diameter tungsten emitter wires on
which carbon whiskers, or dendrites, have been grown.
A high electric field gradient (1010 V/cm) at the tips of
the whiskers produces ionization
48. Spectroscopy
ADVANTAGES
As fragmentation is less, abundance of molecular ions
(M+) is enhanced, hence this method is useful for
relative molecular mass and empirical formula
determination.
DISADVANTAGES
Not suitable for thermally unstable and non volatile
samples.
Sensitivity is les than EI ion source.
No structural information is produced as very little
fragmentation occurs.
52. Spectroscopy
Field Desorption
Also known as offspring of field ionization.
In field desorption method, a multitipped emitter
(made up of tungsten wire with carbon or silicon
whiskers grown on its surface) similar to that used in FI
is used.
The sample solution is deposited on the tip of the
emitter whiskers either by
dipping the emitter into analyte solution or
using a microsyringe.
Then the sample is ionized by applying a high voltage
to the emitter.
53. Spectroscopy
ADVANTAGES
Works well for small organic molecules, low
molecular weight polymers and petrochemical fractions
DISADVA NTAGES
Sensitive to alkali metal contamination.
Sample must be soluble in a solvent.
Not suitable for thermally unstable and non volatile
samples.
Structural information is not obtained as very little
fragmentation occurs.
56. Spectroscopy
Electrospray ionization
Electrospray ionization is a technique used in mass
spectrometry to produce ions from macromolecules
such as proteins, polypeptides and oligonucleotides
having molecular weights of 10,000 Da or more.
The method generates ions from solution of a sample by
creating fine spray of charged droplets.
• A solution of sample is pumped through a fine,
charged stainless steel capillary needle at a rate of few
microlitres/minute.
The needle is maintained at a high electric field
(several kilovolts) with respect to cylindrical electrode.
• The liquid pushes itself out of the capillary as a mist
or aerosol of fine charged droplets.
57. Spectroscopy
These charged droplets are then passed through
desolvating capillary where the solvent is evaporated in
the vacuum and attachment of charge to the analyte
molecules takes place.
Desolvating capillary uses warm nitrogen as nebulising
gas.
The desolvating capillary is maintained under high
pressure.
• As the droplets evaporate the analyte molecules comes
closer together.
58. Spectroscopy
These molecules become unstable as the similarly
and the
charged molecules comes closer together
droplets explode once again. This is referred as
Coulombic fission.
• The process repeats itself until the analyte is free
from solvent and is alone ion.
• The ion then moves to the mass analyzer.
59. Spectroscopy
ADVANTAGES
Most important techniques for analysis of high
molecular weight biomolecules such as polypeptides,
proteins, oligonucleotides and synthetic polymers.
Can be used along with LC and capillary
electrophoresis.
63. Spectroscopy
Plasma Desorption
Plasma desorption produces molecular ions from the
samples coated on a thin foil when a highly energetic
fission fragments from the Californium-252 “blast
through” from the opposite side of the foil.
The fission of Californium-252 nucleus is highly
exothermic and the energy is released.
64. Spectroscopy
When such a high energy fission fragments passes
through the sample foil, extremely rapid localized
heating occurs, producing a temperature in the range of
10000K.
•Consequently, the molecules in this plasma zone are
desorbed.
67. Spectroscopy
Laser Desorption
Laser desorption methods involves interaction of
pulsed laser beam with the sample to produce both
vaporization and ionization.
Laser beam is usually of different wavelengths from far
U.V to far IR depending upon the sample to be
analyzed.
REQUIREMENTS
Laser wavelength must be at absorption wavelength of
the molecule.
In order to avoid decomposition absorbed energy must
be quickly dispersed in the molecules.
68. Spectroscopy
IONIZATION TECHNIQUE:
• Ionization is carried out by two techniques :-
Microprobe techniques
Laser beam is focused to a very small spot on the back
side of a thin metal foil that holds a thin film of sample.
Ions emerge out on the front side from a small cratered
hole in the foil.
Bulk analysis techniques
The technique uses a less focused beam and larger
samples.
The laser beam produces microplasma that consists of
neutral fragments with elementary and fragment ions.
69. Spectroscopy Matrix assisted laser desorption
(MALDI)
Matrix assisted laser desorption is a technique in mass
spectrometry for ionization of biomolecules (polymers
such as proteins, polypeptides and sugars) and synthetic
polymers that are more fragile and form fragments
when ionized by conventional methods.
It consist of two components
1 Matrix : Matrix is used in MALDI to
Absorb the laser energy.
Prevent analyte agglomeration.
Protect analyte from being destroyed by direct laser
beam
70. Spectroscopy
Matrix consists of a crystallized molecules of which the
most commonly used are
Sinapinic acid)
α– cyano cinnamic acid (α –cyano or α–matrix)
Dihydroxy benzoic acid (DHB)
Nicotinic acid
Matrix solution is then mixed with the analyte to be
investigated.
The solution is then spotted in a air tight chamber on
the tip of the sample probe.
71. Spectroscopy With a vacuum pump the air is removed and vacuum is
created which leads to evaporation of the solvent leaving
behind a layer of recrystalized matrix containing analyte
molecules.
2 Laser
The solid mixture is then exposed to pulsed laser beam.
The matrix absorbs the laser energy and transfers some of
this energy to the analyte molecules which results in the
sublimation of sample molecules as ions or the matrix
after
Absorbing the laser energy gets ionized and transfer
part of this charge to the sample molecules and ionize
it.
72. Spectroscopy
When the polymers form cations the cathode is placed
right behind the sample and anode in front of the
sample.
The cations get attracted towards the negatively charged
anode. This acceleration is used to move the ion to the
detector.
When the polymer forms anions the electrodes are
interchanged.
77. Spectroscopy
Fast atom bombardment (FAB) is an ionization
technique used in mass spectrometry in which a beam
of high energy atoms strikes a surface to create ions.
When a beam of high energy ions is used instead of
atoms (as in secondary ion mass spectrometry), the
method is known as liquid secondary ion mass
spectrometry (LSIMS)
80. Spectroscopy
Quadrupole mass analyzer
A quadrupole mass spectrometer contains four parallel
cylindrical rods which can scan or filter sample ions
based on their mass-to-charge ratio.
Opposing rods are connected electrically and a radio
frequency voltage is applied between the pairs of rods.
Ions travel between the rods and only ions with a specific
mass-to-charge ratio will exit the quadrupole; other ions
will collide with the rods.
The desired mass-to-charge ratio can be altered by
changing the applied voltage.
81. Spectroscopy
Triple quadrupole mass spectrometry makes use of the
same technology, but uses a linear series of three
quadrupoles to improve sensitivity and selectivity.
This type of spectrometry is useful when studying
particular ions of interest since it is able to stay tuned to
a single ion for extended periods of time.
83. Spectroscopy
Ion trap mass analyzer
This analyzer employs similar principles as the quadrupole
analyzer mentioned above, it uses an electric field for the
separation of the ions by mass to charge ratios.
The analyzer is made with a ring electrode of a specific voltage and
grounded end cap electrodes.
The ions enter the area between the electrodes through one of the
end caps. After entry, the electric field in the cavity due to the
electrodes causes the ions of certain m/z values to orbit in the
space.
As the radio frequency voltage increases, heavier mass ion orbits
become more stabilized and the light mass ions become less
stabilized, causing them to collide with the wall, and eliminating
the possibility of traveling to and being detected by the detector.
85. Spectroscopy
TOF Analyzers
TOF Analyzers separate ions by time without the use of
an electric or magnetic field.
In a crude sense, TOF is similar to chromatography,
except there is no stationary/ mobile phase, instead the
separation is based on the kinetic energy and velocity of
the ions.
86. Spectroscopy
Ions are accelerated by an electric field of known
strength.This acceleration results in an ion having the
same kinetic energy as any other ion that has the same
charge. The velocity of the ion depends on the mass-to-
charge ratio (heavier ions of the same charge reach
lower speeds)
The time that it subsequently takes for the ion to reach a
detector at a known distance is measured.
This time will depend on the velocity of the ion, and
therefore is a measure of its mass-to-charge ratio. From
this ratio and known experimental parameters, one can
identify the ion.
88. Spectroscopy
Magnetic sector analyzers
Similar to time of flight (TOF) analyzer mentioned
earlier,
In magnetic sector analyzers ions are accelerated
through a flight tube.
Where the ions are separated by charge to mass ratios.
The difference between magnetic sector and TOF is that
a magnetic field is used to separate the ions.
As moving charges enter a magnetic field, the charge is
deflected to a circular motion of a unique radius in a
direction perpendicular to the applied magnetic field