Mass Spectrometry Flashcards
Define atomic number?
The number of protons in one atom
Define atomic mass
The mass of an atom (working to the C12 scale=12.000)
Define molar mass
The mass of one mole (Avogadros number of molecules 6.022x10^23)
measured in Daltons (Da)
Define isotopic mass
Mass of one mole based on a single isotope of each element
Whats a proton
A sub-atomic particle with nominal mass of 1 Da carrying a positive charge
Whats a neutron
A sub-atomic particle with nominal mass of 1 Da carrying no net charge
Whats an electron
A sub-atomic particle of negligable mass when in fact it does have a mass that is needed to be considered when undertaking mass measurements
Whats an ion
A molecule or part of a molecule (or even an atom) with a positive or negative charge
Will always carry a charge depending on structure and how it was charged in the massspectrometer
Whats a cation
A positively charged ion
Whats an anion
A negatively charged ion
Whats a radical
A molecule or part of a molecule with an unpaired electrons
A very reactive species in normal environment
Whats ionisation potential
The energy (voltage and charge,eV) required to ionise a molecule
Whats bond energy
The energy (voltage and charge,eV) required to break a bond
Whats mean free path
Mass spectrometer works at a very high vacuum to help reduce the chance of collisision between species
In the periodic number what is the atomic number and the atomic mass?
- Atomic number= number of protons
- Atomic mass= number of protons + neutrons
What is the basis of mass spec?
A techniques (an instrumental method of analysis)
In which the sample molecules are ionised
And separated according to their mass (m) to charge (z) ratio (m/z)
Z is an integer
The isotopes work on a scale of carbon-12
for C12 and C13 what are they each comprised of?
- 12C is comprised of
o 6 protons
o 6 neutrons
o 6 electrons - 13C is comprised of
o 6 protons
o 7 neutrons
o 6 electrons
What is the natural abundance of carbon isotopes
With this knowledge what is the probability ofa single carbon atom being C13?
- The natural abundance of carbon isotopes is:
o 12C= 100
o 13C= 1.10 - This means that for every 1000 carbon atoms, one expects 11 to be 13C
- The probability of a single carbon atom being 13C is 1.1%
In a compound with 2 carbon atoms, what is the probability of one of thembeing C13?
This is a mass spectrum showing singly charged species including isotope clusters.
What does the base peak show and calculate the number C atoms present
- Relative abundance is 0-100 from largest peak i.e., base peak (644)
- The peak at 645 has an intensity of around 33%. If you divide 33 by 1.1% (C13) then it would suggest there are 30 C atoms present
- The mass to charge scale is largely in integer values, the gap between peaks means there is a distribution to it.The computer generates a straight line in m/z as seen below for morphine
This is the mass spectrum of morphine
what does the base peak represent?
- Consider C in morphine
- 17 carbons
- Integer isotopic mass=285, base peak is representative of whole molecule
- Has C, N and O in its structure
How is the molar mass of morphine calculated?
- Abundance is small for N and O compared C
- C12 also has C13, N14 has N15 and O16 has O18
- The abundance of the heavier isotope is relatively small for N and O compared to C
- C and N have one mass unit different between isotopes whereas O has 2 mass units different
- So don’t need to consider it as are considering one mass unit higher than the isotopic mass
- Only have one N contrasted with 17 C
- Average of isotopes and measure average mass rather than isotopic mass would give molar mass of 285
Theoretically, how many C atoms are there in morphine given all we know about the mass spectra?
These are the masses of sub-atomic particles.
What is the mass of hydrogen?
- Amu= atomic mass units now commonly referred to as Da (don’t need to remember values)
- Must consider mass of electrons in calculations else errors will be significant
- For the H mass it is comprised of a proton + electron mass to give mass of the subatomic particle
Given the mass of the sub-atomic particles, what is the mass of a carbon atom?
When working out the mass of sub-atomic particles, there is a difference of 0.0521502 amu seen. Why is this the case when by definition the atomic mass of C12 is exactly 12?
- Energy is related to mass by Einstein’s famous equation: E=mc2
- This is the binding energy of the nucleus required to separate the neurons and protons
o It is of the order of 8 MeV (8 mega electron volts which is more than electron binding energies)
o Much more than electron binding energies (by about a million)
Here are some accurate masses of some compounds
Distinguise between CO and N2 using accurate mass
There are two definitions for resolution, what are they and what do they mean?
10% valley and FWHM- Full width and half mass
Whats the equation to calculate resolution?
What is the resolution needed to separate CO from N2?
Both have an accurate mass of 28
Other possibilities for mass 28 that can be seen in mass spec are:
C2H4 (ethene) and H2CN+ (protonated formonitrile).
Distinguish C2H4, H2CN+, CO and N2
In ionisation processes in mass spec, how may positive and negative ions forms and how are they displayed?
In mass spec, how are odd electron species formed?
- Written not different as most logical way so e- are closer to molecule and + or – is seen more easily
In the ionisation process, protonation occurs. Complete for the following molecules:
NH3
CH3NH2
CH3COOH
What are the components of a mass spec instrument and what are the typical pressures in each component. Why is this the case?
- Described as having 5 component parts
**o Inlet
o Ionisation source
o Mass analyser
o Detector
o Computer ** - Some mass spectrometers have the ionisation source at atmospheric pressure which is known as atmospheric pressure ionisation that will be covered later
- The mass analyser and detector are always under vacuum to create a mean free path so that the ions don’t collide with neutral molecules and go right to detector
- Inlet could be a chromatograph
- All mass spec have computer
Distinguish C2H4, H2CN+, CO and N2
What are the components of the electromagnetic specturm and what they are measured in?
- Defined in terms of wavelength measured in m ,cm or nm
- Defined by frequency measured in Hertz
- Energy of wavelength/ frequency
- Most energetic rays are gamma and radio waves are the least energetic
How does mass spec relate to the electromagnetic spectrum?
- Mass spectrum works around 8-13 eV which is seen by the arrow
- UV can cause ionisation and cause bonds to break, as occurs in mass spec
- In the EM wavelengths the energy is quantized which means that a molecule or part of molecules absorbs energy or does not. Whereas in mass spec it is a continuum of energy that may be transferred to a molecule
Electron ionisation ion source is how the continuum of energy usually works, explain this equipment and how it functions
- The potential difference between the filament and the trap allows the energy to be transferred from the source
- The electrons want to move across to the relatively positive trap
- At the same time, the filament to the housing has a potential difference which is set at** 70 eV.** What this does is gives the ions an energy that is more than ionisation potential
- Because the e- want to repel each other, kept in narrow beam with a magnet around outsource is put to help focus them together
- Ions produced are repelled into mass analyser where they will be separated based on m/z
- Ionisation potential is around 7-11 eV this is because a certain amount of excess energy is put into each molecule leading to a good probability of ionisation which leads to good sensitivity. however, could still get fragmentation of the molecules
How does a magnetic sector mass analyser work?
- Magnet helps to curve ions around so they will go through a small aperture and into the detector and a signal will be recorded
- To measure ions of different m/z we can gradually increase the field strength of the magnet and therefore detect ions of increasing m/z or vice versa
- This type is rarely used nowadays but is simple to understand
- If it is used its used in isotope ratio mass spec to focus several ions on wider aperture to detect various isotopes of CO and O
Describe how a time of flight mass analyser works
- Time of flight mass analyser is now very popular
- Ions produced inion source region, in this case produced by laser beam
- Ions then repelled into flight tube
- Time for ion squared to reach detection is proportional to m/z (as seen by equation above)
- Light ions travel faster
- Benefits: analyse up to extremely high m/z. gives highest mass range of any mass spec
How do the quadrupole rods in mass analysers work?
- Quadrupole mass analysers when stripped down
- Consists of 4 rods
- Opposing rods linked electrically
- Side to side rods connected together
- Ion beams passes down centre of rods
Explain how the quadrupole mass analyser works
- The ions leave the ion source and take a path through toward detection
- The radio frequency direct cut applied to adjacent poles
- The ion will be attracted or repelled in accordance with RF and offset by direct cut
- Effect of RF:DC means only ions of correct m/z will pass through to detector for any RF:DC ratio
- Ions which are lighter or heavier will not go through central path but will collide with rods and lost from detection
- Only ions with correct m/z for applied RF:DC will get to detector
- Fairly low in cost, very popular
How does an electron multiplier detector work?
- Common detector used
- Ion hitting detector causes emission of electrons
- High V applied to detector surface means e- causes emission or more and more electrons
- So sensitive that even a single ion would be detected
Explain how electrospray ionisation works
- Liquid flow from liquid chromatography column
- Tip at high voltage to respective ground plate
- Around liquid is a Heated stream of hot N gas to evaporate the LC solvent and the solute molecule, the analyte, in liquid drop become concentrated.
- +ve charges come closer before they explode through the drop surface, and you get ions in the gas phase where they pass through an orifice plate into the mass analyser
- Simple protonation often occurs with the soft ionisation techniques forming a protonated ion but with EI you tend to get multiple charges
EWI-MS Yeast alcohol Dehydrogenase
How does Matrix Assisted Laser desorption (MALDI) work?
- The analyte molecules are crystallised in with the matrix molecules which are typically molecules that will absorb laser light energy
- When the laser beam hits the matrix they will absorb energy and evaporate off the surface and transfer charge through to the analyte molecules which are also vaporised before they have a chance to decompose
With this example electron ionisation mass spectrum of amphetamine, it shows that it has a MWof 135. You would expect to see a base peak at 135, however there is one at 44.
With electron ionisation, write the ionisation of a moleculeand what the moleucle is left with and why is this the case?
The effect of electron energy
How ionisation energies differ
With this knowledge of electron energy, explain why amphetamines mass spec doesnt show the molecular ion?
- Molecular ion increases and will eventually plateau
- May have fragment ion which appears higher than ionisation potential as breaking bond in molecule. this difference in energy between molecular and fragment ion is the bond energy
- Where the fragment ion and molecular ion cross over is where the intensity of the fragment= molecular. The fragment ion will continue to increase and increase and may end up that we don’t see a molecular ion. This is seen in amphetamine; the 135 molecular ion is not seen but instead the fragment ion at 44 is seen
- If we reduce the electron energy it will get to a point where the fragment and molecular ion are in equal abundance
How is the molecular ion calculated if there is an unknown compound?
What is the nitrogen rule?
How does chemical ionisation source work?
- Use e- ionisation source but is more gas tight
- Pump in gas that acts as reagent
- Reagent gas ionises molecules
- Lower energy
Based on gasesous phase acid-base reactions
The reagent gas is in a higher abundance than the analyte
Therefore when electrons enter the source they main ionise the reagent gas
The primary reagent ions then undergo secondary ion/molecule reaction where they collect and react with the lower concentration analyte molecules to form product ions
Whats a typical reagent in CI? And how does it work?
What are the principles of fragmentation in MS
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