Mass spectrometry

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.

 The spectra are used to determine The elemental composition of a sample, The masses of molecules, To elucidate the chemical structures of molecules To study isotope abundances Mass spectrometry works by ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios

Mass Spectrometer
A mass spectrometer converts them to gaseous ions so that they can be moved about and manipulated by external electric and magnetic fields The three essential functions of a mass spectrometer, and the associated components, are: 1. The Ion Source A small sample is ionized, usually to cations by loss of an electron. In addition to creating the ions, the ion source form an ion beam. That is, it will provide some degree of focusing, collimation, and acceleration to the ions. 2. The Mass Analyzer The ions are sorted and separated according to their mass and charge. (or more specifically by momentum or energy) This is done using a combination of electric and magnetic fields, sometimes including RF fields 3. The Detector The separated ions are then measured, and the results displayed on a chart.

Mass Spectrometer
Because ions are very reactive and short-lived, their formation and manipulation must be conducted in a vacuum. Residual gases have to be removed within the ion flight path (so that collisions between ions and resident gas does not diffuse the beam), and any waste gases from the ion source can be removed Atmospheric pressure is around 760 torr (mm of mercury). The pressure under which ions may be handled is roughly 10-5 to 10-8 torr (less than a billionth of an atmosphere).

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 and analyzed in a computer

Mass Spectrometer

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 charged repeller 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 inversely proportional to the mass of each ion. (If a charge moves into a magnetic field with direction 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 focused progressively on a detector fixed at the end of a curved tube (also under a high vacuum).

 When a high energy electron collides with a molecule it often ionizes it by knocking away one of the molecular electrons (either bonding or non-bonding). This leaves behind a molecular ion (colored red in the following diagram). Residual energy from the collision may cause the molecular ion to fragment into neutral pieces (colored green) and smaller fragment ions (colored pink and orange). The molecular ion is a radical cation, but the fragment ions may either be radical cations (pink) or carbocations (orange), depending on the nature of the neutral fragment

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

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.p less than 500 C and mol wt less than 1000Da E.g. Electron impact Ionisation (EI) Chemical ionisation (CI) Field Ionisation (FI)

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 105 Da 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)

Another way to classify

A) Hard Sources B)Soft Sources

Hard Sources: Imparts high Energy to the analyte molecules to leave them in a highly excited state 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

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

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 gas phase volatile molecules introduced in a chamber. M+ e- --- M+. + 2e- ( Loss of electron is due to the repulsion between the electrons which 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 forming a continuous beam in a direction perpendicular to the direction of the electron beam. Additional electric fields ( 10 3 to 10 4 V) 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 detection and signal processing, convey structural information about the analyte. The low volatility of heavy mass analytes has precluded the analysis of biological molecules using EI.

Electron Impact Source

High Energy ionisation and hence shows extensive fragmentation Results in a complex mass spectrum This is useful for identification and structure elucidation Advantage: convinient to use, Good sensitivity as it produces high ion currents Extensive fragmentation , so large no. of peaks and hence easy identification

Disadvantage: Sometimes fragmentation is so complete that no molecular ion persits Without molecular ion, mol wt information cannot be obtained Need to volatise the sample which may result in thermal degradation before ionisation Applicable only to mol having mass < 1000Da

 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

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 over the gaseous sample. i.e. the ratio of the conc of the reagent gas to smaple is 103 to 104 This mixture is subjected to electron bombardment (100eV)

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

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

 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

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 metal having 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

Gives a M+ peak Less complicated spectra

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 of M+ 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)

Field Desorption Methods

Multi tip Emitter similar to FI But Electrode is mounted on a probe that can be removed from the sample compartment and can be coated with the solution of the sample or small crystals of solid materials are placed onto the emitter.

This probe is then reinserted into the sample compartment

Slow heating of the emitter then begins, by passing a high current through the emitter, which is maintained at a high potential (e.g. 5 kilovolts). As heating of the emitter continues, low-vapor-pressure materials get desorbed and ionized by alkali metal cation attachment.

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 metal plate Ratio of analyte to matrix is 1:103 to 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 cobalt particles in glycerol could be used as a matrix Used in combination with TOF analyser

MALDI

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 removed from the analyte leaving behind a multi charged analyte molecule

Electron spray Ionisation (ESI)

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

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 generated are accelerated The resultant Ar+ beam is then passed into a chamber containing an argon gas at about 10-3 to 10-4 mm Hg Ar e Ar+ Ar+ + Ar - Ar + Ar + Charge exchange occurs and a beam of fast moving Ar atoms are generated When this beam impinges on the sample, a complex series of ion molecule reactions occur.. Sample ions are thus sputtered from target into the mass analyser Large peptides, aminoglycosides, nucleotides,etc

FAB