Mass Spectrometry

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Mass spectrometry

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• Separate organic compounds of mass <200,000 Dalton (atomic mass and molecular mass)
• Separate proteins on their mass-to-charge ratio

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Uses of Mass Spectrometry

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• ID unknown compounds
• Quantify unknown compounds
• Clarify structures and chemical properties

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Mass spectrometry can be used with

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• GC
• LC
• HPLC

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Advantages of MS

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• Minute quantities of samples
• Compounds can be ID at low concentration

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Applications of MS

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• ID structures of biomolecules
• Sequence biopolymers (proteins, oligosaccharides, etc)
• Drug metabolism
• Forensic analyses; drug abuse
• Analyze environmental pollutants
• Determine age and origin of specimens
• ID and quantitate compounds
• Perform multi-element inorganic analyses

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Mass Spectrometer

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• Measures masses of individual molecules that have been converted to ions (electrically charged)
• Measures mass to charge ratio (m/z)

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Mass to charge ratio

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• Unit: Dalton
• Dalton: 1/12 the mass of a single atom of C^12

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Comparsion of spectrophotometer and MS

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S: Light, Slit, sample, prism, slit, detector

MS: ion source, slit, magnet, slit, detector

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MS Process and components (general parts)

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• Ionization
• Fragmentation
• Separation
• Detection

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MS process

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• Small sample molecules leaks into MS ionization (under high vacuum)
• Molecules are bombarded with high energy electron beam; fragmenting the molecule

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Loss/Gain of electron

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• Loses an electron: becomes positively charged (cation)
• Gains and electron: becomes negative charge (anion)

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Results of electron bombardment

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• Molecules break into characteristic fragments according to molecular structure

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Mass spectrometry Process: Magnetic field and positive fragments

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• Positive fragments accelerated in a vacuum pass tru a magnetic field based on m/z ratio
• The path of the charge molecules bent by magnetic field
• Too small or high momentum will be thrown off the field
• Varying the strength of the magnetic field allows for various m/z to be detected

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MS analogy

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• Ions separated by a magnetic field based on momentum
• Feather will be deflected due to mass
• A bowling ball will continue on its path

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Collision of assorted ion with the detector creates

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an electric current, that is amplified and detected

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The data system records:

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magnitude of electrical signals as a function of m/z and converts it to a mass spectrum

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Sample introduction

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vaporizes samples

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Vacuum system

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reduces collisions between ion and gas molecules

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Ion source

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ionizes analyte gas molecule

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Mass analyzer

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separates ions according to m/z

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Detector

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counts ions

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MS vacuum system

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• Increase the mean free path: the average distance the ions and molecule swill travel before colliding with other ions or molecules
• Ensures predictable and reproducible fragmentation

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Other important use of vacuum system

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• as an insulator against high MS voltages (can damage detector)
• Will not run without pump

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MS ionization methods

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• Electron impact
• Chemical ionization
• Field ionization
• Plasma ionization
• electrospray ionization
• MALDI
• SELDI
• Fast atom bombardment (FAB)

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Ionization method

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• Electron impact (most common)
• Chemical ionization (softer ionization, less fragmentation)

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Electron Impact

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• "hard" ionization method- fragmentation can sometimes be too extensive
• Bombards sample with electrons

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Electrion impact process

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• Electron (70eV) strives a neutral molecule
• Knocks out a second electron
• Produces a molecular ion and energy
• Positive charged repelled by repeller so they are pushed to the detector
• Passes the first lens (magnetic) of increasing negative charge
• Accelerates so fast it collides into analyzer

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Chemical Ionization

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• EI performed in the presence of a gas
• Causes reduced fragmentation and increase production of molecular ion
• Gas and sample enter at the same time
• Donation of a charged species from gas to analyte for a positively charge ion

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Chemical Ionization limitations

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• line production dependent on the gas and gas pressure
• Reference spectra not as extensive as EI

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ESI (Electrospray Ionization Theory)

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• Large droplets explode into smaller droplet until it enters the gas phase as an ion
• Creates variably charged ions
• Number of positive charges a molecule can hold is related to the number of basic sites on the molecule

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MALDI-Matrix assisted laser desoprtion/Ionization

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• Absorption of laser energy by a matrix compound
• First analyte formed into a solid solution
• Analyte molecules separated creating a homogenous solution; matrix excitation
• Liquid solvents removed before they are dried

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TOF

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Time of flight

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MALDI-TOF

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• Ions from the sample are focused
• time required to reach the detector is a nonlinear function of the mass
• larger ions require more time to reach the detector

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SELDI

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Analyze proteins captured on the surface of the protein chip array or by biomolecules covalently bound to the array by means of specific surface chemistries

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Protein chip process

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• Proteins are non-specifically adhered to the chip, salts are washed away
• Energy absorbed molecules mediate ionization of protein
• Incorporate proteins into crystals
• Chip placed in analyzer
• A laser is fired at the chip: ionization and desorption
• Detector records TOF and derives the mass

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Mass analyzer

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• Filter
• Resolves different mass fragments and separates them
• Uncharged molecules/fragments pumped away
• Molecular ions and fragments accelerated at the same energy

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Mass analyzer types

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• Magnetic
• electrostatic
• time of flight
• quadrupole mass filter
• quadrupole ion storage

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Ion Trap process

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• At fixed intervals sample is ionized and trapped
• Due to positive potential
• reducing positive potential they will drop out of the trap
• Ions are ejected in order of increasing m/z ratio

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Quadrupole

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• Four rods each carry a voltage in pairs
• DC filed is applied to 2 rods and RF to the other
• Strength and freq of RF detemines what ions pass tru dependent on mass
• Only ions of proper m/z can traverse the filter

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Quadrupole potentials

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• Combinations of potentials is applied to the rods varied with time to separate various ions

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Quadrupole scanning

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Spectrum is obtained by monitoring the ions passing through the filter as the rod voltage varies

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MS detectors

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Work by producing an electronic signal when struck by an ion of m/z

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MS hitograms

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• Results from detector converted into a histogram
• Most intense peak: base peak
• All other peaks based on base peak
• Highest MW peak is the parent molecule

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Atomic absorption

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• Used for measuring heavy metals
• Most atoms in ground state
• Small sample size
• Sensitive
• Energy absorbed corresponds to concentration of analyte in the sample

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Atomic absorption spectrophotometry

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• Sample is weighed and dissolved (strong acid)
• Resulting solution is sprayed into the flame and atomized
• light of appropriate wavelength is shone through the flame, some light is absorbed by the atoms
• Amount of light absorbed is proportional to concentration of element

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AA with graphite furance

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• Electro-thermal heating to increase sensitivity
• Sample deposited in graphite
• with gas to protect tube from oxidation as temp rises and to remove gaseous by products
• Sample volatilizied and excited
• Concentration in the tube 1000x more than that in a flame