Mass Spectrometry

Question 1

mass spectroscopy

Answer 1

Involves particles, not radiation. It measures the massed charge ratio of the ion.

Question 2

Parts of a mass spec 1

Answer 2

The mass analyser: TOF, Quadrupol, FT-ICR, Sectors Orbitrap, ion trap

Question 3

Parts of a mass spec 2

Answer 3

Dectector:

Electron multiplier, Microchannel plate, ion-photon detectors FT-ICR Orbitrap

Question 4

What are the steps involved in mass measurement

Answer 4

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)

Question 5

What is the purpose of the mass analyser and detector being inside a VACCUM

Answer 5

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.

Question 6

What is positive and negative ion modes?

Answer 6

positive: molecule has ADDITIONAL proton.
negative: molecule has a proton less.

Question 7

Isotopes

Answer 7

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.

Question 8

The resolution

Answer 8

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%.

Question 9

resolution

Answer 9

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

Question 10

Accuracy and precision

Answer 10

High precision hits close together but does not hit the target so accuracy is low.

Needs to be both accurate and precise

Question 11

Electrospray ionisation (ESI) electrospray occurs in the first few cm of mass spec.

Answer 11

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)

Question 12

List some ionisation techniques

Answer 12

Electrospray ionisation (ESI)

Matrix-assisted laser desorption/ionisation (MALDI)

Electron Ionisation (EI),

Chemical Ionisation (CI),

Fast-Atom Bombardment (FAB)

Question 13

What are the advantages of Electrospray ionisation (ESI)

Answer 13

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)

Question 14

How to get the mass from a ESI-MS spec.

Answer 14

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)

Question 15

What does the convoluted spectra show?

Answer 15

From m/z calculations, one peak is produced showing the exact mass of protein

Question 16

How does MALDI work.

Answer 16

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)

Question 17

MALDI ionisation

Answer 17

target spots; tiny amount of sample (0.5microliters) mixed with matrix.

sample can be reanalyse sample.

Question 18

What properties must the MALDI matrix have?

Answer 18

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.

Question 19

How fast are lasers?

Answer 19

2kHz; very very fast; 1500 samples in 2 hours

Question 20

Applications of MALDI

Answer 20

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.

Question 21

Advantages of MALDI?

Answer 21

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)

Question 22

Disadvantages of MALDI

Answer 22

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.