10-08-2012, 12:35 PM
Mass spectrophotometer
[attachment=30923]
INTRODUCTION AND PRINCIPLE
Molecules are first ionized then two types of ions are generated- parental molecule ions and fragment ions.
These ions will have similar charge but different mass, i.e. they will differ in m/e ratio.
These ions are accelerated through an electric field in vacuum.
According to m/e ratio ions are deflected towards magnetic plate and will be separated.
Separated ions are then subjected to detector, which detect the ion beam current for each ion type. This will give a mass spectrum.
Using this mass spectrum molecular weight and structure of compound can be determined.
Sample introduction
Sample must be introduced into vacuum (to minimize ionization error)
It must be in vapor form (to facilitate complete ionization of sample)
Sample can be introduced by capillary infusion.
Sample ionization
Electron ionization method: sample is heated to obtain in vapor form, then bombarded with electron beam (70 ev).
This will result into ionization and fragmentation of sample molecule.
Suitable for- Mol wt upto 400D
For heat stable molecules
Electrospray:
Aqueous or organic solutions are forced through a narrow needle, which is kept at high potential (3.5 KV).
This high potential will causes ionization as the sample is nebulized.
As the size o droplet decreases the charge density increases.
These droplets of similar charges will repel each other.
These are passed through a high vacuum (several torr) in this condition the thin film of solvent also get evaporated.
Advantages: can be used for heat labile compounds.
It result into multiple charges which is helpful for macromolecule analysis.
Also suitable method for liquid samples.
MALDI: Matrix Assisted Laser Desorption Ionization
A matrix is used to evaporate and ionize the sample.
Sample is crystallized with matrix and then exposed to Uv and laser light beam.
The matrix will absorb light and get evaporated at the same time the molecules of sample also reaches in vapor.
Matrix help in ionization also.
Matrix-2,5 dihydroxybenzoic acid and cyno-4 hydroxycinnamic acid and sinapinic acid.
It is very useful technique for analysis of nucleic acid, peptides and proteins means biological macromolecules.
Mass analyzers
Magnetic sector mass analyzer: J. J. hompson 1897.
Megnetic sectors will separate ions according to m/e ratio.
A 1-10 kV electric field accelerates the ions through megnetic sector.
The charged ions get deflected in an arc. The radius of the arc depends upon momentum of the ion.
Greater the momentum larger will be radius of the arc.
Momentum of ion depends upon mass and charge of ion and megnetic field strength.
As the charge on particle and megnetic field strength is same for all particles so they will separate according to mass.
These ions are focussed on photographic plate or slit of fixed radius.
Detectors
Faraday cup: a change in charge on metal plate causes flow of electron. Less sensitive but simple method
Electron multiplier method: signal is amplified and then detected so it has better resolution
Photomultiplier conversion dynode: here multiplied electrons are sent to phosphorus screen it emits photons, which are detected by photomultiplier. Here vacuum is used that reduces chances of error due to contamination and makes it more sensitive.
[attachment=30923]
INTRODUCTION AND PRINCIPLE
Molecules are first ionized then two types of ions are generated- parental molecule ions and fragment ions.
These ions will have similar charge but different mass, i.e. they will differ in m/e ratio.
These ions are accelerated through an electric field in vacuum.
According to m/e ratio ions are deflected towards magnetic plate and will be separated.
Separated ions are then subjected to detector, which detect the ion beam current for each ion type. This will give a mass spectrum.
Using this mass spectrum molecular weight and structure of compound can be determined.
Sample introduction
Sample must be introduced into vacuum (to minimize ionization error)
It must be in vapor form (to facilitate complete ionization of sample)
Sample can be introduced by capillary infusion.
Sample ionization
Electron ionization method: sample is heated to obtain in vapor form, then bombarded with electron beam (70 ev).
This will result into ionization and fragmentation of sample molecule.
Suitable for- Mol wt upto 400D
For heat stable molecules
Electrospray:
Aqueous or organic solutions are forced through a narrow needle, which is kept at high potential (3.5 KV).
This high potential will causes ionization as the sample is nebulized.
As the size o droplet decreases the charge density increases.
These droplets of similar charges will repel each other.
These are passed through a high vacuum (several torr) in this condition the thin film of solvent also get evaporated.
Advantages: can be used for heat labile compounds.
It result into multiple charges which is helpful for macromolecule analysis.
Also suitable method for liquid samples.
MALDI: Matrix Assisted Laser Desorption Ionization
A matrix is used to evaporate and ionize the sample.
Sample is crystallized with matrix and then exposed to Uv and laser light beam.
The matrix will absorb light and get evaporated at the same time the molecules of sample also reaches in vapor.
Matrix help in ionization also.
Matrix-2,5 dihydroxybenzoic acid and cyno-4 hydroxycinnamic acid and sinapinic acid.
It is very useful technique for analysis of nucleic acid, peptides and proteins means biological macromolecules.
Mass analyzers
Magnetic sector mass analyzer: J. J. hompson 1897.
Megnetic sectors will separate ions according to m/e ratio.
A 1-10 kV electric field accelerates the ions through megnetic sector.
The charged ions get deflected in an arc. The radius of the arc depends upon momentum of the ion.
Greater the momentum larger will be radius of the arc.
Momentum of ion depends upon mass and charge of ion and megnetic field strength.
As the charge on particle and megnetic field strength is same for all particles so they will separate according to mass.
These ions are focussed on photographic plate or slit of fixed radius.
Detectors
Faraday cup: a change in charge on metal plate causes flow of electron. Less sensitive but simple method
Electron multiplier method: signal is amplified and then detected so it has better resolution
Photomultiplier conversion dynode: here multiplied electrons are sent to phosphorus screen it emits photons, which are detected by photomultiplier. Here vacuum is used that reduces chances of error due to contamination and makes it more sensitive.