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MADD > Mass Spectrometry > Flashcards

Mass Spectrometry Flashcards

1
Q

What does MS measure? (x2)

A

Molecular mass and provides structural information of organic compounds/peptides/proteins

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2
Q

Where is MS used? (x4)

A

Pharmaceutical analysis, Biomolecule characterisation, Environmental analysis and Forensic/Clinical analysis

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3
Q

Two methods of sample introduction?

A
  1. Directly into the ionisation source
  2. Undergo chromatography first e.g. HPLC, GC - separates the sample into a series of components which enter the ionisation source sequentially
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4
Q

What does MALDI stand for?

A

Matrix Assisted Laser Desorption/Ionisation

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5
Q

What is MALDI commonly used for?

A

Analysis of synthetic and natural polymers, proteins and peptides

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6
Q

Up to what MW can MALDI operate?

A

200,000 Daltons

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7
Q

How is MALDI set up?

A

Sample is dissolved in a volatile solvent and co-crystallised in a matrix material (e.g. sinapinic acid) to absorb the laser and both are applied to the MALDI plate

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8
Q

Wavelength of MALDI laser and nature of beam?

A

337 nm; pulsed laser beam is directed at the matrix and the sample

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9
Q

What happens in response to the pulsed laser beam in MALDI?

A

Matrix absorbs light energy from the laser, causing the rapid heating of the crystals - matrix crystals vaporise into the gas phase taking the sample with them

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10
Q

In MALDI, what ions are formed and how?

A

Singly charged ions formed by proton transfer from matrix to sample molecules

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11
Q

Why is MALDI known as a “soft ionisation technique”?

A

Very little energy is transferred to the analyte, allowing ionisation of the sample without fragmentation

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12
Q

How are desorbed ions measured in MALDI?

A

With a time of flight mass analyser

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13
Q

What is the purpose of electrospray ionisation (ESI)?

A

To transfer ions in solution into gaseous ions at atmospheric pressure (LC/MS)

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14
Q

What compounds is ESI typically used for?

A

Thermally labile and high molecular weight compounds

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15
Q

How is the sample introduced in ESI?

A

Sample flows through a stainless steel capillary to which a high voltage (3 - 4 kV) is applied to the tip, creating a large potential between the inlet needle and cone

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16
Q

What happens to the sample emerging from the tip in ESI?

A

It is dispersed into an aerosol of highly charged droplets

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17
Q

In ESI, what happens when a positive potential is applied to the tip of the capillary?

A

Positive ions will accumulate at the surface of the liquid and will move away from the capillary tip towards the sample inlet cone which is maintained at a lower voltage

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18
Q

In ESI, what molecules are usually analysed under positive ionisation conditions?

A

Proteins and peptides

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19
Q

(ESI) What happens to the cone of positively charged droplets as they move towards the sample inlet cone?

A

They are subjected to heat or heated nitrogen gas, causing the solvent to evaporate and droplet size to reduce

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20
Q

How do droplets release charged sample ions in ESI?

A

Droplets shrink until they reach a point where the surface tension can no longer sustain the charge they are holding (known as the Rayleigh limit), causing a coulombic explosion and break down of the droplet

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21
Q

In ESI, what do samples with a molecular weight of up to 1,200 Da give rise to?

A

Singly charged molecular ions, usually in the form (M+H)+ in positive ionisation mode

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22
Q

In ESI, what do samples with a molecular weight greater than 1,200 Da give rise to?

A

Multiply charged ions such as (M+nH)n+ in positive ionisation mode

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23
Q

Advantages of soft ionisation techniques?

A

Little or no fragmentation, makes it easy to obtain peptide mass fingerprint

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24
Q

Disadvantages of soft ionisation techniques?

A

Does not facilitate protein identification so often need to do tandem mass spectrometry (e.g. collision-induced dissociation)

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25
Q

Advantages and disadvantages for MALDI?

A

Advantages: Very quick and easy to use, low cost, high accuracy
Disadvantages: Less sensitive

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26
Q

What does electron impact ionisation (EI) lead to?

A

Extensive fragmentation so that the molecular ion is not observed for many compounds

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27
Q

What is fragmentation useful for in EI however?

A

Provides structural information for interpreting unknown spectra

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28
Q

What happens to gas-phase molecules after they enter the ion source in EI?

A

A beam of electrons moves through the ion source - negative charge distorts the electron cloud forming a radical cation that frequently fragments into a fragment ion and a radical

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29
Q

What is the filament in EI made of and how is the number of electrons emitted controlled?

A

Tungsten or Rhenium; amount of current controls number of electrons emitted by the filament

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30
Q

Advantages of EI?

A

High ionisation efficiency and sensitivity, providing fingerprint of the compound (fragmentation aids identification of compounds); does not require much sample;

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31
Q

Disadvantages of EI?

A

Molecules must be volatile and thermally stable; useful mass range is quite low (<1000 Da)

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32
Q

What mass analysers are commonly used?

A

Time of Flight, Ion traps and Magnetic sectors

33
Q

What does selection of a mass analyser depend upon?

A

The resolution, mass range, scan rate and detection limits

34
Q

Advantages of the TOF mass analyser?

A

Fixed voltages and does not require a magnetic field

35
Q

Disadvantages of the TOF mass analyser?

A

Poor mass resolution

36
Q

How does the TOF mass analyser work?

A

At the source, ions are formed by a rapid ionisation pulse - these are accelerated into the flight tube by an electric field (2 - 25 kV) applied between the backing plate and acceleration grid

37
Q

How are ions separated in TOF?

A

Since ions are accelerated across the same distance by the same force, they have the same kinetic energy; lighter ions will travel faster to reach the detector

38
Q

What does the time delay from the formation of the ions to the time time they reach the detector depend on in TOF?

A
  1. Length of the drift region
  2. Mass/charge ratio of ions
  3. Acceleration voltage at the source
39
Q

In TOF, how do we compensate for minor differences in kinetic energies of ions with the same m/z?

A

Using an ion reflector

40
Q

In TOF, how do we compensate for the spatiotemporal differences of ion formation?

A

Using delayed extraction, i.e. allow ions to expand within the source, introduce a time delay and extract all the ions at the same time

41
Q

What does the Quadrupole mass analyser consist of and how is it set up?

A

4 parallel rods equally spaced around a central axis - rods opposite each other are connected with the same DC and RF voltage

42
Q

Advantages of Quadrupole mass analyser?

A

Compact size, fast scan rate, high transmission efficiency

43
Q

Disadvantages of Quadrupole mass analyser?

A

Limited to a unit m/z resolution

44
Q

How does the Quadrupole separate ions?

A

RF and DC voltages produce an oscillating electric field that functions as a bandpass filter to transmit selected m/z value (high and low pass filters allowing ions within a set m/z range to pass through the centre of the quadrupole

45
Q

What are the components of an ion trap?

A

Two end traps and a ring electrode

46
Q

How are ions trapped in the ion trap?

A

Combination of RF and DC voltages applied to ring and end cap electrodes, respectively, creates a quadrupole electric field which traps ions in a potential energy well - inside the trap, ions take on the oscillating frequency that is related to their m/z ratio

47
Q

How is mass resolution increased in the ion trap mass analyser?

A

A small amount of Helium is added as a bath gas, which collides with ions thereby dampening their motion and increasing trapping efficiency

48
Q

How are ions ejected from the ion trap?

A

As DC and RF voltages are increased, frequency of ion oscillations increases such that it becomes destabilised and is ejected from the trap

49
Q

What are the advantages of using a magnetic sector mass analyser?

A

Higher resolution and greater mass range compared to quadrupole instrumnets

50
Q

Disadvantages of using a magnetic sector mass analyser?

A

Require a larger vacuum pump and often scan more slowly

51
Q

How are ions accelerated in a magnetic sector?

A

Into the magnetic field through slits

52
Q

What happens to ions when they encounter the magnetic field?

A

They are deflected towards the detectors, in a direction perpendicular to the magnetic field so ions follow a circular trajectory with radius R

53
Q

(Magnetic sector) What is the radius of the arc dependent on?

A

The momentum and charge of the ion, as well as the magnetic field strength

54
Q

How is the m/z determined in the magnetic sector mass analyser?

A

m/z = [(r^2)*(B^2)]/2V - only 1 m/z value will satisfy this equation for a given radius, magnetic field strength and acceleration voltage - mass spectrum is scanned by changing the magnetic field and the acceleration voltage to transmit different m/z values to the detector

55
Q

How do detectors generate a signal from incident ions?

A

Either by generation secondary electrons (which are then further amplified) or by inducing an electric current generated by a moving charge

56
Q

What do most detectors currently used do?

A

Amplify the ion signal using a collector similar to a photo/electron multiplier

57
Q

What do electron multipliers consist of and how do they work?

A

They consist of electrodes called DYNODES; when an ion hits the first dynode, two electrons are emitted that are accelerated to the second dynode, where each causes two more electrons to be ejected… and so on… cascade of electrons

58
Q

What are the two types of set up for electron multipliers?

A

Either a series of dynodes maintained at increasing potentials, OR, a channel multiplier (shaped like a horn), where amplification occurs through repeated collisions with the dynode surface

59
Q

What does 1 Dalton correspond to?

A

1/12th of the mass of a single 12C atom

60
Q

In spectra, what is observed when there are isotopes present?

A

Two or more molecular ions peaks

61
Q

How can the molecular ion be identified on a MS?

A

Usually the highest m/z peak in the spectrum (but this could be an impurity or an isotope of the molecular ion)

62
Q

If the peak is the molecular ion, what is the next highest peak?

A

The fragmented ion having a lost a neutral fragment

63
Q

What is the molecular mass of most compounds?

A

Even

64
Q

What does the nitrogen rule state?

A

Any compound with an odd number of nitrogens will have an odd molecular mass (even number of nitrogens = even molecular mass)

65
Q

How can we rule out the molecular ion peak?

A

If a compound does not have any nitrogen but gives an odd peak

66
Q

How can the molecular ion be verified?

A

By chemical ionisation - as the ionisation energy is very low, the molecular ion intensity is higher and the adduct ion (MH+*) is observed at m/z M + 1

67
Q

What is sigma bond cleavage?

A

Removal of an electron from a sigma bond weakens it, such that one fragment gets the remaining electron and is a neutral radical (R*) while the other is a charged species (R+)

68
Q

What is a-cleavage caused by?

A

When an electron from a bond to an atom adjacent to the charge site pairs up with the radical

69
Q

When does inductive cleavage occur?

A

When the charged site induces a pair of electrons to migrate from an adjacent bond or atom, breaking a sigma bond

70
Q

What happens in the McLafferty rearrangement?

A

A group (typically a H) is transferred from another place in the molecule to the site of ionisation

71
Q

What feature of rearrangements is important?

A

These reactions are more complex and involve the making and breaking of bonds - they are thermodynamically favourable because they require less energy

72
Q

How are rearranged fragments identified?

A

Because they are observed as odd electron ions with an even m/z ratio

73
Q

Factors to consider in fragmentation of the molecular ion?

A
  1. Energy of the molecular ion and fragments formed from it
  2. Stability of bonds in the ions
  3. Steric factors
  4. Stability of the formed ions or neutral particles
74
Q

Stevenson’s rule?

A

When a bond is cleaved the fragment with the lowest ionisation potential will carry the charge

75
Q

Which fragmentation processes will occur with higher relative abundances?

A

Those that lead to the most stable cations and radicals

76
Q

Where are the peaks found for primary, secondary and tertiary alcohols?

A

m/z 31, 45 and 59, respectively

77
Q

In which cases is further fragmentation necessary?

A

ESI and MALDI

78
Q

Three ways of performing tandem mass spectrometry?

A

Collision-induced dissociation (CID), Electron-transfer dissociation (ETD) or Electron-capture dissociation (ECD)

MADD (5 decks)

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