Mass Spectrum_L1 Flashcards
the definition of mass spectrometry
the instrument which is used to define the covalent structure of a molecule by ionizing separating, detecting the molecular and fragmental ions according to the mass to charge ratios.
what are the advantages of using mass spectrometry over other instruments?
(1) sensitive, the input amount is not large
(2) do not need high-quality purification, crude complex can be detected using the technique
what are the three basic parts consisted in the mass spectrometry?
(1) ion source: the biological sample is converted to gas phase
(2) ion analyser: the analyser separates the fragmented or molecular ions based on various approaches
(3) detector: quantify the detected information by using the mass spectrum graph
the meaning of intensity on the y-axis of the mass spectrum
The intensity of each ion is measured in percentage relative to the most abundant compound, therefore, it is the relative abundance rather than absolute abundance
three different ionization methods
(1) Electron Impact (EI - Hard)
– small molecules, 1-1000 Da
(2)Electrospray Ionization (ES or ESI - Soft)
– peptides, oligosaccharides, proteins, greater than 500,000 Da
(3)Matrix Assisted Laser Desorption Ionisation (MALDI - Soft)
– peptides, proteins, DNA, up to 500,000 Da
ES/ESI and MALDI are both good at handling large-molecular compounds
What is the difference between the hard and soft ionization approaches?
The hard ionization refers the compound ionization event induced by excessive high energy which causes the generation of fragmented ions, wherease the fragmented ions are not present in the soft ionization since the energy is lower and only changes the molecule into gas phase
the principle of electron impact
(1) Sample introduced into source by heating it from a probe tip until it evaporates or from an on-line gas chromatograph, the large molecules are not likely to be turned into gas phase by heating, this is one of the reasons why the suitable targets of EI are mainly small molecules.
(2) Gas phase sample is bombarded with electrons coming from rhenium or tungsten filament (energy = 70 eV), electrons tend to come from the heated metals.
(3) Ionization occurs by loss of an electron to give M+. (radical cation), radical cations are singly charged.
(4) Most of the molecular ions decompose into fragments (70 eV»_space; 5 eV bonds) via uni-molecular reactions, the bombarded electron energy is far greater than the bond energy, excessive energy causes the formation of fragments. A singly charged electron repulses against the orbital electron present in the sample, repulsion force drives the loss of an electron, the sample is thus positively charged. Since the electron size is small, the probability of hitting to the biological sample is extremely small.
write an equation for EI-MS
M + e = M.+(singly charged and in radical form) + 2e
describe arrangement of main set-up in EI
(1) metal filament: generate electrons by heating
(2) ion repeller: postively charged to repel the gas-phase ion to the ion exit slit
(3) magnent: focus the electron beam, increase the probability of hitting onto the biological sample, biolohical sample is exposed to the beam for cations production
(4) extraction, focusing and source slit are three components when the ions exit from the sample, the three components tightly control the electron movement
preparation and principle of MALDI
(1) sample and matrix are applied to the target metal for natural drying-out and crystalisation
(2) Sample is embedded in a low molecular weight UV absorbing “crystalline” matrix which is chosen to have an absorption maximum near the wavelength of the pulsed laser that is used to ionise the sample
(3) The matrix absorbs the laser pulse and enough energy is transferred to the sample to ionise it, energy is firstly absorbed by the matrix and then transmitted to the sample under the vaccum to generate gas-phase ions. These ions are either positively or negatively charged.
(4) Process not well understood - believed to be similar to “flash evaporation”
(5) Depending on the ions of interest, the positively charged magnent is placed at the back to repel the cations for them to enter the analyser field.
lasers for MALDI
(1) nitrogen-gas UV laser
(2) solid-state UV laser: more favourable due to longer wavelength
common matrix for MALDI
(1) Alpha-cyano-4-hydroxycinnamin acid (alpha CHCA) for peptides and proteins: the matrix has maximum absorbance and emission from the laser
(2) 2,5-dihydroxybenzoic acid(2,5-DHB)for carbohydrates: matrices contain aromatic rings which absorb energy and transmit to the biological sample
How to improve the resolution of the MALDI?
Delayed extraction, the technique infers that the pulse of ions is kept in the source for a short time after the laser pulse in order to allow time for ions formed deep in the matrix to emerge and “catch up” with those formed near the surface, as the crystal is a three-dimensional structure, the order of absorbing the laser is different for molecules at the front and at the back. Ions of the same mass coming from the target have different speeds. This is due to uneven energy distribution (energy spread) when the ions are formed by the laser pulse, depending on where in the crystal the ion is formed, the quality of mass spectrometry is interfered
How does the delayed extraction ensure the ions to be in same velocity when entering the analyser?
The ions are generated from the matrix with different energy absorbed, those migrating faster are used to be buried in position which is closer to the matrix surafce. A field is applied to accelerate the migrating ions, ions travelling in faster speed are more away from the matrix and are exerted by less force, thereby less acceleration, vice versa. Eventually, all the ions are moving in the same speed theoretically.
processes/principles of ES and ESI
(1)Atmospheric pressure ionisation technique
(2)Sample introduced into source in solution through a narrow glass capillary coated with gold at the tip
(3)A high voltage (3-4 kV) is applied to the tip
Sample emerging from the tip is dispersed as an aerosol of highly charged droplets, force is imposed on the needle to push the droplets out from the needle, as the sample passes from the electrode towards the mass spectrometry
(4) Process aided by a drying gas (nitrogen) that flows around the outside of the capillary. As the solvent evaporates the droplets become unstable due to high surface charge - “explode” into smaller droplets, peptides are forced to be closer in the droplet
(5) Eventually “naked” ions whose charge corresponds to the charge of the original molecule at the pH of the experiment and the amino acid composition. These ions of interest drift into the analyser because of the voltage gradient
