mass spectrometry.ppt 1

8/12/2019 Mass Spectrometry.ppt 1

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Mass spectrometry


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Mass spectrometry

Mass spectrometry is a powerful analytical technique used to

quantify known materials,

to identify unknown compounds within a sample,

and to elucidate the structure and chemical properties of different

molecules.

The complete process involves the conversion of the sample into

gaseous ions, with or without fragmentation, which are then

characterized by their mass to charge ratios (m/z) and relative

abundances.


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The spectra are used to determine

The elemental composition of a sample,

The masses of molecules,

To elucidate the chemical structures of moleculesTo study isotope abundances

Mass spectrometry works by ionizing chemical compounds to generatecharged molecules or molecule fragments and measuringtheir mass-to-charge ratios
http://en.wikipedia.org/wiki/Moleculehttp://en.wikipedia.org/wiki/Mass-to-charge_ratiohttp://en.wikipedia.org/wiki/Mass-to-charge_ratiohttp://en.wikipedia.org/wiki/Mass-to-charge_ratiohttp://en.wikipedia.org/wiki/Mass-to-charge_ratiohttp://en.wikipedia.org/wiki/Mass-to-charge_ratiohttp://en.wikipedia.org/wiki/Mass-to-charge_ratiohttp://en.wikipedia.org/wiki/Molecule


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Mass Spectrometer

A mass spectrometer converts them to gaseous ions so that they can bemoved about and manipulated by external electric and magnetic fields

The three essential functions of a mass spectrometer, and the associatedcomponents, are:

1. The Ion SourceA small sample is ionized, usually to cations by loss of

an electron. In addition to creating the ions, the ion source form an ionbeam. That is, it will provide some degree of focusing, collimation, andacceleration to the ions.

2. The Mass AnalyzerThe ions are sorted and separated according totheir mass and charge. (or more specifically by momentum or energy) Thisis done using a combination of electric and magnetic fields, sometimes

including RF fields

3. The DetectorThe separated ions are then measured, and the resultsdisplayed on a chart.


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Mass Spectrometer

Because ions are very reactive and short-lived, their formation and manipulation mustbe conducted in a vacuum.

Residual gases have to be removed within the ion flight path (so that collisionsbetween ions and resident gas does not diffuse the beam), and any waste gasesfrom the ion source can be removed

Atmospheric pressure is around 760 torr (mm of mercury). The pressure under whichions may be handled is roughly 10-5 to 10-8 torr (less than a billionth of anatmosphere).

Each of the three tasks listed above may be accomplished in different ways.

In one common procedure, ionization is effected by a high energy beam of electrons,

and ion separation is achieved by accelerating and focusing the ions in a beam,which is then bent by an external magnetic field.

The ions are then detected electronically and the resulting information is stored andanalyzed in a computer


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Mass Spectrometer


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Ion source. Here molecules of the sample (black dots) are bombarded by electrons(light blue lines) issuing from a heated filament.

This is called an EI(electron-impact) source.

Gases and volatile liquid samples are allowed to leak into the ion source from a reservoir

Non-volatile solids and liquids may be introduced directly.

Cations formed by the electron bombardment (red dots) are pushed away by a chargedrepeller plate (anions are attracted to it), and accelerated toward other electrodes,

having slits through which the ions pass as a beam.

Some of these ions fragment into smaller cations and neutral fragments.

A perpendicular magnetic field deflects the ion beam in an arc whose radius is inverselyproportional to the mass of each ion. (If a charge moves into a magnetic fieldwithdirection perpendicular to the field, it will follow a circular path. )

Lighter ions are deflected more than heavier ions.

By varying the strength of the magnetic field, ions of different mass can be focusedprogressively on a detector fixed at the end of a curved tube (also under a highvacuum).
http://hyperphysics.phy-astr.gsu.edu/hbase/magnetic/magfie.htmlhttp://hyperphysics.phy-astr.gsu.edu/hbase/magnetic/magfie.html


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When a high energy electron collides with a molecule it often ionizesit by knocking away one of the molecular electrons (either bondingor non-bonding).

This leaves behind a molecular ion(colored red in the followingdiagram).

Residual energy from the collision may cause the molecular ion tofragment into neutral pieces (colored green) and smaller fragment

ions(colored pink and orange).

The molecular ion is a radical cation, but the fragment ions mayeither be radical cations (pink) or carbocations (orange), dependingon the nature of the neutral fragment


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The Nature of Mass Spectra

Mass spectra is a line spectrum

Each line represents an ion having a specific mass-to-charge ratio (m/z)

and the length of the bar indicates the relative abundance of the ion.

The most intense ion is assigned an abundance of 100, and it is

referred to as the base peak. Most of the ions formed in a mass spectrometer have a single

charge, so the m/z value is equivalent to mass itself.

The highest-mass ion in a spectrum is normally considered to be the

molecular ion (followed by ions containing heavier isotopes) ,and lower-mass

ions are fragments from the molecular ion, assuming the sample is a

single pure compound


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Ion sources

Function to produce gaseous analyte ions

2 types of sources: a) Gas phase Sources

b) Desorption sources

Gas Phase sources:

Here the sample is first vapourised and then ionised

So they can be used only to ionise thermally stable compounds that have b.pless than 500 C and mol wt less than 1000Da

E.g. Electron impact Ionisation (EI)Chemical ionisation (CI)

Field Ionisation (FI)


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Desorption Sources

Here the solid or liquid state sample is converted directly to gaseous ions

Applicable to non volatile and thermally stable samples

Does not require volatisation of the compounds and hence applicable to

analytes having mass as large as 105Da

E.g. Field desorption (FD)

Matrix assisted desorption ionisation((MALDI)

Fast atom bombardment (FAB)

Plasma Desorption (PD)

Thermospray ionisation(TIS

Secondary ion mass spectrometry(SIMS)


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Another way to classify

A) Hard Sources

B)Soft Sources

Hard Sources:

Imparts high Energyto the analyte molecules to leave them in a highly excitedstate

This energy surges through the molecule rupturing the bonds and producing

the fragment ions that have m/z ratio less than the molecular ion ( less mol

wt fragments)

So the mass spectrum has many peaks corresponding to small fragments

E.G Electron Impact ionisation


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Soft Sources

Causes little fragmentation

Mass spectrum consits of molecular ion peak and only a few other

ion peaks

E.g Chemical Ionisation

Useful as they supply accurate information about molecular mass of

the analyte molecule or molecules

Eg Chemical ionisation


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Electron Impact Source First the sample is brought to temp high enough to produce the molecular vapour

Here the electrons emitted through thermionic emission by passing current across a

Tungsten or Rhenium filament

They are then accelerated by applying 70 V between the filament and the anode

Now they have kinetic energies sufficient to knock-off valence electrons from gasphase volatile molecules introduced in a chamber.

M+ e- ---M+. + 2e- ( Loss of electron is due to the repulsion between the electronswhich bombard the molecule vapours and the electron cloud of the molecule itself)

The radical cations formed are extracted from the chamber by a repeller plate forminga continuous beam in a direction perpendicular to the direction of the electron beam.

Additional electric fields ( 10 3to 10 4V) external to the chamber are employed to aid

extraction and present a focused ion beam at the entrance to the mass analyzer.

The ionization process often gives rise to fragment ions which, following detectionand signal processing, convey structural information about the analyte.

The low volatility of heavy mass analytes has precluded the analysis of biologicalmolecules using EI.


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Isotope peaks: peaks may also occur at m/z ration greater than

molecular ion.. This is attributable to ions having the same chemical

formula but different isotopic composition.The size of these peaks

depends on relative natural abundance of these isotopes

Collision product peaks :Peaks seen at high m/z ratio than mol ion


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2) Chemical Ionisation Sources

Here the gaseous atoms of the sample are ionised by collision with

the ions produced by the electron bombardment of the excess of the

reagent gas

Here the reactant gas is admitted in several thousand excess overthe gaseous sample. i.e. the ratio of the conc of the reagent gas to

smaple is 103to 104

This mixture is subjected to electron bombardment (100eV)

The primary ionisation occurs of the reagent gas as its conc is very

high


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Chemical Ionisation Sources

Reagent ions so formed undergo rapid reaction with their own

neutral species to form steady state ion plasma This in turn interacts with the molecules of the sample to ionise them

One of the most common reagent gas is methane

CH4+ e -CH4+.

+ 2e CH4

+.CH3+ + H.

CH4+.+ CH4 --CH5

+ + CH3.

CH3+ + CH4---C2H5

+ + H2


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These ions react with analyte:

Most common ions (M+1)+ and (M-1)+, which are known as Quassi

molecular ion

Spectra of M+ion is very weak

Other reagent gases which are used are Isobutane or ammonia

Ion plasma with a high proton affinity causes little or no

fragmentation and gives uncomplicated spectrum of sample with an

intense quasi molecular ion

These spectra are easier to interprete


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Field Ionisation Sources and spectra

Low energy method ( Soft Method)

Here the ions are formed under the influence of a large positive

electric field

Such fields are produced by applying high voltages ( 10 to 20 kV) to

specially formed emitters consisting of numerous fine tips of metalhaving diameter less than 1 m on a tungsten wire

The high electric field gradient between the sample molecule and

the metal results in a loss of electron by a molecule to the anode

The ions formed are repelled by the anode and accelerated to MS

analyser FI emitters are mounted 0.5 to 2 mm from cathode which also

serves as a slit

Lesser sensitive than EI


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Gives a M+peak

Less complicated spectra


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Desorption Sources

Applicable for non volatile and thermally unstable compounds

Also for thermally labile biochemical samples

Here energy in various forms is introduced in such a way that it

causes direct formation of gaseous ions

As a consequence, spectra are generally simplified and consits ofM+ ions or protonated (M+1)+ ion

E.g.Field desorption methods (FD)

Matrix Assisted Laser Desorption Ionisation (MALDI)

Electro spray ionisation (ESI) Fast Atom Bombardment (FAB)


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Field Desorption Methods

Multi tip Emitter similar to FI

But Electrode is mounted on a probe that can be removed from thesample compartment and can be coated with the solution of thesample or small crystals of solid materials are placed onto theemitter.

This probe is then reinserted into the sample compartment

Slow heating of the emitter then begins, by passing a high currentthrough the emitter, which is maintained at a high potential (e.g. 5

kilovolts).

As heating of the emitter continues, low-vapor-pressure materialsget desorbed and ionized by alkali metal cation attachment.


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Matrix-Assisted Laser Desorption/Ionization

(MALDI):

Soft ionisation tech A low conc of analyte is dispersed in a liquid or a solid matrix

This is deposited on on the end of the stainless steel probe or metalplate

Ratio of analyte to matrix is 1:103to 1: 105

This plate is then placed in a vacuum chamber A laser beam is focussed onto the sample

The matrix strogly absorbs the laser radiation

The energy gets transferred from matrix to the analyte

The matrix and analyte are then desorbed and ionised to create the

ion plume Lasers include nitrogen (337nm) or CO2

Application : analysis of biomolecules like DNA, proteins, peptides

30 nm cobaltparticles in glycerol could be used as a matrix

Used in combination with TOF analyser
http://en.wikipedia.org/wiki/Cobalthttp://en.wikipedia.org/wiki/Glycerolhttp://en.wikipedia.org/wiki/Glycerolhttp://en.wikipedia.org/wiki/Cobalt


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MALDI


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Electron spray Ionisation (ESI)

Takes place under atmospheric Pressure and temp

Sample solution is pumped through stainless steel capillary needle

The needle is maintained at several kilovolts with respect to a cylindrical

electrode which surround the needle

Results in charged spray of fine droplets passing from needle into

desolvating capillary

Droplets become smaller and smaller as a result of evaporation of solvent ,

and their charge density becomes greater

When surface tension can no longer support the charge the droplet gets

torn apart into smaller droplets

These small droplets can repeat the process until all the solvent is removedfrom the analyte leaving behind a multi charged analyte molecule


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Electron spray Ionisation (ESI)


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Advantages of ESI

Little fragmentation of lage thermally labile mol as there is little extra energy

retained in the analyte molecule upon ionisation

The ions formed are multiply charged , so m/z values are small enough to

make them detectable with a quadrupole instrument

ESI is readily adapted to direct the sample introduction from HPLC columns

Used for analysing biomolecules of very high mol wt like polypeptides,

proteins, oligonucleotides

Also characterisation of inorganic spp and synthetic polymers is possible


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Fast atom Bombardment (FAB) Here the ionisation of analyte from solid/solution state is achieved by

a beam of fast, highly energetic neutral atoms of the xenon or argon

gas The sample in condensed state is in a viscous solution matrix like

glycerol, thioglycerols,sroen ethers etc

Argon gas is first ionised by a hot filamnet, and the ions generatedare accelerated

The resultant Ar+beam is then passed into a chamber containing anargon gas at about 10-3to 10-4 mm Hg

Are Ar+

Ar+ + Ar -Ar + Ar +

Charge exchange occurs and a beam of fast moving Ar atoms aregenerated

When this beam impinges on the sample, a complex series of ionmolecule reactions occur.. Sample ions are thus sputtered fromtarget into the mass analyser

Large peptides, aminoglycosides, nucleotides,etc


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FAB

mass spectrometry.ppt 1

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