Mass spectroscopy

principle

separates ionized particles by using different ratios of their charges to their respective masses in an electric/ magnetic field

molecules fragmented into different pieces

only ions are detected

Three main components of MS

Ion source : sample molecules are ionized

mass analyser : ions sorted and seperated a=ccording to atomic mass

doctor : seperated ions are measured and displayed on mass spectrum

vaporization

ionization

acceleration

separation in magnetic field

detector

applications

v specific and sensitive for determine structure of molecules

impurity characterisation

ligand-protein interaction characterisation

enzyme inhibition assays

Electron impact

Hard method used for small molecules

Electron impact ionisation method

sample evaporated and introduced into instrument

gas phase sample - bombarded with electrons coming from heated filament

molecule ionized and shattered into fragments

ions sent to analyzer

electron impact advantages

well established

fragmentation libraries

suitable for insoluble and/or non polar samples

electron impact disadvantages

no molecular ion identification

need volatile sample

must maintain thermal stability

Electron spray ionization

Soft method for proteins and peptides up to 200 kDa

Matrix assisted laser desorption

For peptides and proteins up to 500kDa

Method of electron spray ionization

- liquid enters - high voltage applied + sample ejected from capillary and sprayed forming charged droplets

- droplets evaporate due to counter current of heated gas, surface charge density increases

- columb explosion separates ions

- size of droplets -> nm level, ion droplets turn into gas phase

electron spray advantages

molecular ion

suitable for high mass +/or thermally labile compounds

easy to operate

ESI disadvantages

sample must be polar

no fragmentation

need solubility in polar solvents

Nominal mass

integer mass of the species with the most abundant isotope of each of the constituent atoms

Monoisotopic mass

Exact mass of most abundant naturally occuring isotope relative to c12

Magnetic sector analyser

Ion source

magnet and analyser tube with ion collector

Matrix-assisted laser desorption ionization (MALDI)

soft method for peptides, proteins, DNA + molecules up to 500kD

MALDI method

analyte molecules embedded in a crystalline matrix composed of a low MW organic species

mix is struck with short intense laser pulse

rapid heating -> sublimation and expansion into gas phase

MALDI advantages

molecular ion

high mass + thermally labile compounds

easy to operate

MALDI disadvantages

no fragment library

quantitation difficult

background interference

MS vs NMR

MS:

more sensitive

allows for analysis of larger molecules

more difficult sample prep

not quantitative

name 4 types of analyzers

magnetic sector

quadruple

ion trap

time of flight

Ion Trap

electric field separates ions based on m/z ratio

- ions trapped in ring electrode

- heavier ions - more stable and travel to detector

- lighter ions - less stable - collide with wall + not detected

magnetic sector analyser

separates ions in a magnetic field based on momentum and charge of ion

lighter ions - more deflected (low m/z)

heavier ions - less deflected (high m/z)

quadrupole analyzer

4 cylindrical rods set parallel to each other

Ions are separated based on how stable their paths are in the oscillating electric fields applied to the rods.

cheap, small, good for ions with low energy

time of flight

separation based on kinetic energy and velocity of the ions

measures how long it takes ion to hit the end of tube

large molecules travel slower

collisionally activated dissociation

technique used to induce fragmentation of selected ions in the gaseous phase

puts energy into ions to induce

ionisation potential

energy needed to remove the weakest bound electron

higher energy electrons are most vulnerable

nitrogen rule

molecular ions of an analyte with odd no. of nitrogen will have odd m/z value

MI with even no. of N = even m/z value