Mass spectrometry

(about the theory and applications)

By

Dr. Ahmed M. Metwaly

Objectives

• Definition

• Applications

• Time line

• Theory

• Instrument

• Terminology

Definition • Mass spectrometry is an analytical technique that measures

the mass-to-charge ratio of charged particles

Applications This technique is applicable in:

• Identifying molecular Wight.

• Detecting fragmentation pattern.

• Determining the molecular formula (HR-Ms).

• Determining the isotopic distribution.

• Protein sequence (Ms-Ms)

(Weighs molecules)

Theory • The physics behind mass spectrometry is that a charged

particle passing through a magnetic field is deflected along a circular path on a radius that is proportional to the mass to charge ratio, m/e.

• Different chemicals have different masses.

• In the first phase of the mass spectrometry these masses may be vaporized (turned into gas) and ionized (broken down) into ions.

• An electric field accelerates the ions to a high speed.

• The accelerated ions directed into a magnetic field which applies a

force to each ion perpendicular to the plane.

• This force deflects the ions (makes them curve instead of traveling in a straight line) to varying degrees depending on their mass\charge ratio.

• Lighter ions get deflected more than the heavier ions. (due to Newton's second law of motion. The acceleration of a particle is inversely proportional to its mass).

• The detector measures the deflection of each resulting ion beam.

• Most stable ions appears as higher peaks due to higher abundance.

Instrumentation

Mass spectrometers consist of three basic parts: an ion source, a mass analyzer, and a detector system.

The stages within the mass spectrometer are:

• Production of ions from the sample (ionization).

• Separation of ions with different masses (deflection).

• Detection of the number of ions of each mass produced.

• Collection of data to generate the mass spectrum.

Ion source • The ion source is the part of the mass spectrometer that

ionizes the material under analysis (the analyte), usually to cations by loss of an electron, because ions are easier to manipulate than neutral molecules. The ions are then transported by magnetic or electric fields to the mass analyzer.

• Electron ionization and chemical ionization are used for gases and vapors. In chemical ionization sources, the analyte is ionized by chemical ion-molecule reactions during collisions in the source. Two techniques often used with liquid and solid biological samples include electrospray ionization, matrix-assisted laser desorption/ionization (MALDI) and fast atom bombardment (FAB).

Mass analyzer

• Mass analyzers separate or resolve the ions formed in the ionisation source of the mass spectrometer according to their mass-to-charge ratio (m/z). All mass spectrometers are based on dynamics of charged particles in electric and magnetic fields in vacuum.

• The mass spectrum calculated according to compounds mass-to-charge ratio (m/z) because;

The position of peaks on the spectrum depending on;

1. Direct proportional to mass (heavier molecules deflected less and reach detector first)

2. Inverse proportional to the charge (although most molecules have one charge) in case of a molecule with 2 charges, it will deflected more (the deflection force takes place over the charges) and it will reach the detector late.

Detection and recording of sample

ions • The detector records the charge induced or current

produced when an ion passes by or hits a surface, amplifies it and the signal is then transmitted to the data system where it is recorded in the form of mass spectra .

Methods of sample ionization

(Ionization techniques): Hard ionization technique:

• a lot of energy involved.

• extensive fragmentation of analyte molecules.

• Electron Impact (EI) most common, uses an electron beam (70 eV electrons) to ionize samples.

Soft ionization techniques:

• Less energy is used to ionize the analyte molecule.

• Less fragmentation occurs (softer on the molecule).

Soft ionization methods include the

following:

• Chemical Ionisation (CI) most common.

• Electrospray Ionisation (ESI).

• Fast Atom Bombardment (FAB).

• Matrix Assisted Laser Desorption Ionisation (MALDI).

Electron Impact (EI):

• This method is used for ionization of low molecular weight

compounds.

• In an electron impact mass spectrometer, a high energy beam

of electrons is used to displace (dislodge) an electron from the

organic molecule in the gas phase to form a radical cation

known as the molecular ion or parent ion.

• The molecular ion fragmented to give other positively

charged smaller ions (fragment ions) and a radical.

M + e- M + 2 e- m1+ + m2

Ionization Fragmentation

Electron Impact (EI):

Advantages of EI: • Sensitive.

• Its ability to produces a considerable number of fragment ions which help in identification of the compound.

• Fragmentation provides “fingerprint”.

Disadvantages of EI:

• Molecular ion peak often eliminated.

• Sample volatilization necessary.

• Not suitable for thermo labile compounds.

Terminology:

• Molecular ion (M+): The ion obtained by the loss of an electron from the molecule and represent the molecular weight of the compound.

• Base peak: The most intense peak in the MS, it is assigned a value of 100% intensity and the intensities of other peaks are reported as percentages of the base peak.

• Radical cation: The positive charged species with an odd number of electrons.

• Fragment ions: The lighter cations formed by the decomposition of the molecular ion. These often

correspond to stable carbcations.

Summary

• Definition

• Applications

• Time line

• Theory

• Instrument

• Terminology