maas spectroscopy
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MASS SPECTROMETRY
Index
Introduction
Principle
General Instrumentation
=> Sample Introduction (Inlet)
{A} Ionization
1. Electron Ionization
2. Electro Spray
3. Matrix – Assisted Laser Desorption/ Ionization (MALDI)
{B} Mass Analyzers
1. Magnetic Sector Analyzer
2. The Quadrupole Analyzer
3. Time of Flight Analyzer
{C} Detectors
1. Faraday cup
2. Electron Multiplier
3. Photomultiplier Conversion Dynode
Applications of Mass Spectrometery
Reference
• MS is powerful analytical
technique.
• Identify unknown compounds.
• Quantify known materials.
• Elucidation of structural and
chemical properties.
•Requires minute quantities
(<pg).
• High sensitivity , selectivity
and specificity – provides
valuable information in various
branches of science.
Introduction
Mass Spectrometer is an instrumental technique in which, sample is
converted to rapidly moving positive ions by electron bombardment and
charged particles are separated according to their masses.
PRINCIPLE
Principal based on:-
1. First event is to ionize the molecule.
2. Ions move into mass analyzer where the ions are differentiated according to their mass to charge ( m/z ) ratio.
3. The differentiated ion beams now fall onto the detector , which measures the ion beam current.
General Insrumentation
A sample introduction system (inlet system)
1. An ion source,
2. A mass analyzer and
3. A detector
Sample Introduction
To introduce a sample two things must be achieved here:-
• One is that the sample must be introduced into vacuum
• Secondly , the sample must be vaporized prior to ionization, so that proper
ionization take place.
To introduce sample one approach is by placing a sample on a probe, which is than
inserted through a vacuum lock into the ionization region of MS.
Another method of introducing the sample is capillary infusion.
This delivers small quantities of sample to the ionization chamber without
disturbing the vacuum.
The capillary can be a column from a gas /liquid chromatography.
Ionization
Ionization means placing a charge on an otherwise neutral molecule.
Several different methods :-
1.Electron Ionization
2.Electro spray Ionization
3.Matrix- Assisted Laser Desorption/ Ionization (MALDI)
Electron Impact (EI)
•The original mass spectrometry (MS) ionization method
•The most widely used
•Used for ionization and fragmentation the sample molecules before mass analysis
•Firstly the sample is vaporized and then enters into the ion source
•A beam of electrons help the sample be impacted with sufficient
energy to ionize the molecule
Electrospray Ionization (ESI)
• Soft ionization technique, desorption ionization method
• Proteins, peptides, and other biological macromolecules
• No fragmentation of the macromolecules into smaller charged particles; turning the
macromolecule being ionized into small droplets
• Large mass molecules are detected in the small mass range of the instrument
•As the m/z value increases, the number of protons attached to the molecular ion decreases
Matrix-Assisted Laser Desorption Ionization (MALDI)
• A laser-based soft ionization method
• Important for protein analysis.
• Ionization occurs with bombarding the sample with laser light
• The sample is embedded in a chemical matrix (UV absorbant)
• The matrix makes the production of intact gas-phase ions from large, nonvolatile, and
thermally decomposed compounds such as proteins, oligonucleotides, synthetic polymers
easy
• The matrix absorbs the laser light energy thus small amount is vaporized
Mass Analyzer
Mass Analyzer is a region of the mass spectrometer is used to separate
ions within a selected range of mass – to – Charge (M/Z) ratio.
To separate ions different method used:-
1.Magnetic Sector Analyzer
2.The Quadrupole Analyzer
3.Time of Flight Analyzer
Magnetic Sector Analyzer
• J.J. Thompson (1897), built the mass spectrometer , used a magnet to measure the
M/Z value .
• Magnetic Sector instrument used today evolved from this concept.
• Separates ions in a magnetic field according to the momentum & charge of the ion.
• An electric field (1-10 kv) accelerates ions
from source region intomagnetic sector.
• When it reaches magnetic field ,the ion
beam bent in an arc by the
magnetic field.
• Greater the momentum of ions, larger
their arc radius.
• Instrument have a set of slites at a fixed radius, transmitt a single M/Z to detector.
Time-of-Flight Analyzer
• Used with the MALDI technique for analysis of large biomolecules
• All ions have same kinetic energy.
• Thus having same kinetic energy leads different velocities depending on their masses
• Masses affect their arrival time at the detector, smaller ions will reach the detector first
due to they have greater velocity and vise versa.
• Since, time of reaching the detector is of essense in this analyzer, hence name given.
• Arrival time of an ion at the detector is based upon mass, charge & kinetic energy of the
ion.
Quadrupole Analyzer
• An electric field accelarates ions
into the quadrupole analyzer.
• Consist of 4 rodes/ electrodes
arranged across from each other.
• Ions made to travels through
the Quadrupole.
• They get filtered according to their M/Z ratio.
• M/Z ratio is based upon the radio frequency & direct current voltages applied to
these electrodes.
• These voltage produce an oscillating electric field ,transmitt ions according to their
M/Z value.
MS Detector
Detector generates a signal current from incident ions by generating secondary
electrones which are further amplified.
Most commenly used detectors are :-
1.Faraday Cup
2.Electron Multiplier
3.Photomultiplier Conversion Dynode
Faraday Cup
• A change in charge on a metal plate results in a flow of electrons. The flow creates a
current. This is the concept on which faraday cup detector operates.
• When a single ion strike the surface of a dynode in the faraday cup, it results in
ejection of several electrons.
• This ejection of electrons induces a current in the cup.
• A small amplification may also results because of secondary electrons.
Electron Multiplier
•Concept same as faraday cup.
•Only difference is that faraday cup uses a
single dynode, an electron multiplier uses a
series of dynodes maintained at successively
higher potential.
•Electrons released by the first dynode when
the ion strike on it are dragged to second
dynode because it has a higher potential.
•These primary electrons strike the second
dynode with a force and the collision releases
even more secondary electrons which too are
dragged and so on.
•Typical amplification of an electron multiplier
is one million.
Photomultiplier
•Similar to electron multiplier in that the ion strike and resulting in the emission of
electrons.
•These electrons now made to strike on a phosphorus screen. This screen releases
photons when electron strike it.
•This photons are now detected by a photomultiplier.
•Photomultiplier tube housed in vacuum , removes the possibility of any
contamination from the internal environment.
•It has a higher life for this reason this detector are becoming more popular
Applications of MS
• Protein characterization.
• Peptide mass finger printing and protein identification.
• Applications in virology.
• Sequencing peptide and oligonucleotides.
• Determination of higher order protein structure.
• Analysis of biological noncovalant complexes.
REFERENCES
Avinash Upadhyay, Himalaya publishing house, Biophysical Chemistry, Fourth edition, chapter 15, Mass spectrometry, pg no. 576-585.
http://www.google.com
http://www.wikipedia.com
.
Thank you
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