Mass Spectrometry Flashcards
mass spectroscopy
Involves particles, not radiation. It measures the massed charge ratio of the ion.
Parts of a mass spec 1
The mass analyser: TOF, Quadrupol, FT-ICR, Sectors Orbitrap, ion trap
Parts of a mass spec 2
Dectector:
Electron multiplier, Microchannel plate, ion-photon detectors FT-ICR Orbitrap
What are the steps involved in mass measurement
1) Sample Introduction (Chromatography, direct injection, capillary electrophoresis)
2) Ionisation (MALDI) it needs so it can move in an electric field
3) Mass Analyser
4) Detector
5) Data Acquisition (computer)
What is the purpose of the mass analyser and detector being inside a VACCUM
Ultra high vacuum less than 10-7 mbar Achieved by turbo pumps. Pump 500 meters of air per second; very powerful
This is needed to ensure that secondary ions do not collide with background gases on their way to the detector (i.e. the mean free path of gas molecules within the detector must be large compared to the size of the instrument) Mean free path (average distance travelled by ion before it collides with an air molecule= 1/ gas number density (N) x collision cross section between ion and molecule
It also prevents surface contamination by adsorption of background gas particles during measurement.
What is positive and negative ion modes?
positive: molecule has ADDITIONAL proton.
negative: molecule has a proton less.
Isotopes
each atom will have a natural isotopic ratio due to the difference in the number of neutrons which therefore affect its mass
C12 and C13; mass difference/ mass of one proton = 1.007
C12 has greater abundance than C13
Cl 35 and Cl37
The isotope pattern is dependent on the composition and number of atoms
Analysis of these patterns the charge state (eg ion+2)can be found; it is the difference between two peaks; double charge ion means the mass/charge ratio becomes half a dalton.
The resolution
resolution measures of the ability to distinguish two peaks of slightly different mass-to-charge ratios ΔM, in a mass spectrum.
R= M/ΔM
where M was mass of the second peak and ΔM is the resolving power
A greater resolution means there is a greater separation of peaks.
ΔM= width of the peak measured at a specified fraction of the peak height, for example 0.5%, 5%, 10% or 50%.
resolution
resolution of 500, can easily separate two peaks at the mass of 50
A mass of 1000, at the same resolution, the peaks cannot be separated. This important to see masses that are very similar but not identical, ie Carbon isotrope 19, 20, 13
200 and lower resolution is very low; start to separate at 2000
Typically, 10,000-500,000 used
Accuracy and precision
High precision hits close together but does not hit the target so accuracy is low.
Needs to be both accurate and precise
Electrospray ionisation (ESI) electrospray occurs in the first few cm of mass spec.
most used in biological mass spec
Sample somewhere in liquid; inject sample. Sample comes through a capillary somewhere attached to the Voltage (metal)
Apply needle through needle to mass spec at 2000-500 volts
When you sample this in he capillary, there is a lot of charge droplets, at some point droplets emerge from tip. (the spray) Capillaries very small.
The droplets are very highly charged on the surface; the liquid is removed but the charge will always be there.
There is so much charge on a very small droplet, can cause droplet to explode which form smaller droplets. This repeats until single molecules left with charge.
All the neural ions and removed solvent should stick to the outside of the mass spec (not used)
List some ionisation techniques
Electrospray ionisation (ESI)
Matrix-assisted laser desorption/ionisation (MALDI)
Electron Ionisation (EI),
Chemical Ionisation (CI),
Fast-Atom Bombardment (FAB)
What are the advantages of Electrospray ionisation (ESI)
It comes straight from solution so it is a very soft ionisation technique
Easy to couple SEPARATION techniques such as nano-LC.
osmotically charged ions.
proteins can be kept in their negative form (because it is out of solution)
How to get the mass from a ESI-MS spec.
Space between peaks is the difference of one charge; m/z value; calculate mass.
nH = number of protons
mn = (mass+number of charges) /charge (n)
What does the convoluted spectra show?
From m/z calculations, one peak is produced showing the exact mass of protein
How does MALDI work.
We put analyte together with the matrix.
Matrix excited by a laser, absorbs photon energy in its AROMATIC RING and so transfers energy/PHENOLIC photon to the analyte all in the GAS state
The transfer minimises aggregation between analyte molecules themselves.
Only matrix is excited; analyte would be destroyed if targeted by the laser.
produces single charged ions from polymers/large biomolecules of high masses
Not sensitive to contaminants
High sensitivity at attomole level
High throughput analysis (lots of samples processed per day)
MALDI ionisation
target spots; tiny amount of sample (0.5microliters) mixed with matrix.
sample can be reanalyse sample.
What properties must the MALDI matrix have?
1) Absorb strongly at Laser wavelength.
2) Have low sublimation temperature.
3) Have good mixing and solvent compatibility with analyte.
4) Have ability to participate in photochemical reaction
They are all aromaic. dithranol has three rings.
How fast are lasers?
2kHz; very very fast; 1500 samples in 2 hours
Applications of MALDI
It can use double or single charged ions preferentially
Used for polymers weight distribution; each peak is a different polymer chain; chain length determined.
Polymers abundant in many samples; overpowers any other molecules.
Advantages of MALDI?
Relatively gentle ionization technique.
Very high MW species can be ionized.
Molecule need not be volatile.
Very easy to get femtomole sensitivity.
Usually 1-3 charge states, even for very high MW species. (easier to analyse)
Positive or negative ions from same spot. (this can be repeated as long as their is sample left)
Disadvantages of MALDI
MALDI matrix cluster ions obscure low m/z (<600) range.
Analyte must have very low vapor pressure. (if it disapears in the matrix it cant be analysed)
Pulsed nature of source limits compatibility with many mass analyzers.
Coupling MALDI with chromatography is very difficult.
Analytes that absorb laser light can be problematic as they will be destroyed.
