Mass Spectrometry Flashcards
What type of techniques does GCMS combine?
Chromatographic and spectral
where is the column directly inserted into?
MS ionization chamber
Why do all interface devices contain a heat source?
To prevent analyte condensation within the transfer line
What must this heat not cause?
Thermal decomposition of analyte
Name 3 advantages of direct capillary interface?
Low cost, no dead volume, no selectivity
What are 3 disadvantages of direct capillary interface?
limits flow range that column can use, limits ID of column that can be used, part of column is ‘lost’ - serves as a flow restrictor
volatile substances can be ionised by electron impact ionisation by interacting with what?
an electron beam generated by a heated filament in the ion
why are internal standards necessary in GC?
due to the need for normalisation due to volume changes
What does adding a fixed concentration of IS to each standard mean for calibration?
calibration can be carried out using peak area ratio and thus ignores changes in injection volume
What is the injection volume of LC?
10-50uL
Why is the precision better in LC than GC?
a fixed volume loop is filled with sample and only this amount goes on every time
why is it common in both LC and GC to extract samples prior to analysis?
due to a need for preconcentration or because the sample matrix is not appropriate for chromatographic system
What is commonly done prior to extraction to compensate for the sample being lost?
addition of an internal standard
What is the advantage of considering peak area ratios instead of peak area?
normalisation is achieved and precision and accuracy are better
In LC and GC, why must the internal standard be separated from the analyte?
For an internal standard to be of use otherwise you could not distinguish area representing each
why is this one of the most difficult tasks for a chromatographer?
because the internal standard must have similair chromatographic properties as the analyte and be able to be detected in the same way
what is deuterium
a stable isotope of hydrogen depicted as 2H, has a mass of 2
what are 2 important characteristics a deuterated standard should have
should coelute with compound to be quantified, should also contain enough mass to show a signal outside the natural mass distribution of the analyte
what are the 2 most common interfaces used in HPLC?
electrospray ionisation and atmospheric pressure chemical ionisation
what is the more recently used interface in HPLC?
Atmospheric pressure photo ionisation
in electrospray ionisation, what flow rate is the analyte typically intoduced at?
1uL min-1
The analyte solution flow passes through the electrospray needle that has high potential difference applied to it - what does this cause?
This forces the spraying of charged droplets from the needle with a surface charge of the same polarity to the charge on the needle
what are the droplets repelled towards from the needle?
source sampling cone on counter electrode
What occurs as the droples traverse the space between the needle tip and cone?
solvent evaporation
What happens to the droplet as solvent evaporation occurs?
it shrinks until it reaches the point that surface tension can no longer sustain the charge (rayleigh limit)
This causes a ‘coulombic explosion’ to occur, what does this do to the droplet?
it is ripped apart, causing smaller droplets which can repeat the process
what makes this method of ionisation very soft?
very little residual energy is retained by the analyte upon ionisation
what is a major disadvantage to electrospray ionisation?
very little fragmentation is produced
how might this be overcome?
with the use of tandem mass spectrometric techniques such as MS/MS
What is Atmospheric pressure chemical ionisation suitable/not suitable for?
suitable for low mass compounds, not suitable for thermally liable compounds.
In APCI, what is the analyte solution introduced to?
a pneumatic nebulizer
What is the analyte then desolvated in?
heated quartz tube
What does the analyte then interact with? What does this create?
The corona discharge - creates ions
what does the corona discharge replace in Cl?
the electron filament
what does this produce?
Primary ions; N2 and N4 by electron ionisation
What do these primary ions collide with to form secondary reactant gas ions; H3O and (H2O)nH+?
the vaporized solvent
What causes the formation of analyte ions
reactant gas ions colliding with analyte
What does high frequency of collisions result in? 2
High ionisation efficiency and thermalisation of the analyte ions
What will the resulting spectra look like?
predominently molecular species and adduct ions with very little fragmentation
What do the ions enter after forming? is this similair to ESI?
the pumping and focussing stage in a similair way as ESI
In Atmospheric pressire photo ionisation (APPI), what does the discharge lamp generate?
photons in a narrow range of ionization energies
why is the range of energies carefully chosen?
to ionize as many analyte molecules as possible while minimising ionisation of solvent molecules
out of electrospray and APPI, which is more complicated in terms of ion production?
APPI
What are the 2 ways that radical ions can be produced in APPI?
direcctly or via an intermediate using a dopant
what is the fundamental process in photoionisation?
the absorption of a high energy photon by the molecule and subsequent ejection of an electron
In direct APPI, this process occurs for the analyte molecule, forming what?
molecular radical cation M+
The analyte radical cation can be detected as M+ or……?
it can react with surrounding molecules and be detected as another ion
What is the most common reaction?
The abstraction of a hydrogen atom from the abundent solvent
what does this reaction form?
form the stable [M+H]+ cation, which is usually the observed ion
In dopant APPI (or photoionization-induced APCI) a quantity of photoionizable molecules are introduced to what?
the sample stream to create a source of charged carriers
Give 2 examples of a photoionizable molecule?
toluene or acetone
what can be used to give the same effect?
A photoionizable solvent
What do the dopant or solvent ions react with? and via what reactions?
neutral analyte molecules via proton transfer or charge exchange reactions
How can APPI produce negative ions? 2
By creating alot of thermal electrons from dopant or solvent ionization, or by photons striking metal surfaces in the ionization source
How do mobile phase additives affect the analysis? (2)
purity and composition
what do polymers, including proteins and DNA, form adducts with?
inorganic salts
What does this lead to?
complicated mass spectra and a broad distribution of multiply-charged sodium, potassium and chloride adducts
Reagents, chemicals, sample preparation and post column additives always present what risk to the analysis?
contamination
Why are alkali ions, plasticizers and surfactants particularly problematic?
As they are widespread and interfere strongly with LC-MS by causing higher background noise and the formation of adducts
give 4 examples of additives used in LC-MS?
Formic acid, Acetic acid, ammonium formate, ammonium acetate, ammonium hydroxide solution
what are small organic acids commonly added to influence?
analyte ionization
Name a reason for formic and acetic acid’s widespread use in terms of separation?
separation benefits in terms of retention and/or peak shape under acidic conditions
why is this?
because any silanol activity is suppressed
Most mass spectrometric measurements are done in positive ion mode, which is accomplished by the addition of a proton to form the molecular ion [M+H]+. Why can formic and acetic acid readily fulfil this purpose?
They have necessary acidity and volatility to provide an excess of cations for this purpose
why do mobile phases for HPLC of proteins and peptides usually contain trifluoroacetic acid? 2
to contol pH and improve peak shape and resolution
How does it enhance retention and improve peak shape?
By ion-pairing with the peptide and reducing silanol interactions
what does TFA’s high surface tension prevent?
efficient spray formation
TFA ions in gas phase ion-pair with peptide’s basic group, what does this then suppress?
Their ionisation and reduces the MS signal
If TFA cant be avoided, what can be added to reduce its effects?
small organic acids known as triple blends
what might the addition of a triple blend improve (1) and weaken (2)?
signal improves, resolution and sensitivity poorer
why might it be necessary to use more neutral conditions in some circumstances?
may be because the analytes are sensitive to acids or do not exhibit optimal resolution at low pH
Give 2 examples of volatile salts that can be used when acids are not suitable?
ammonium formate or ammonium acetate
Their use is much more complicaed than acids, what are the 3 issues they may cause?
Limited solubility of salts in organic solvents, changing pH during a gradient run, the acidic pH provided by salts permits both + and - ion mode detection
the main issue concerns the solubility. Why is its lack of solubility in acetonitrile a problem?
acetonitrile is the organic solvent of choice for most separations
What is used to stabilise the special blend containing 0.1% w/v of ammonium acetate in acetonitrile?
acid
what are the 3 desirable effects of this?
salt is kept in a solution, solution is stable against decomposition and keeps the pH in the mildly acid range
What is tandem mass spectrometry
hyphenated technique with more than one mass spectrometer used together
What is the first MS used for?
to separate or isolate molecular ions of various analytes in a mixture
What is the second MS called and used for?
its known as collision cell and molecular ions are fragmented
What is the 3rd MS used for?
Scans for fragments of the molecular ion
What is the first quadrouple (MS1) in a tandem situation usually equipped with?
a soft ionisation technique (eg electrospray
ionisation)
Not much fragmentation takes place here, resulting in what?
the production of only the molecular ion or protonated molecular ions
When the ions are passed to the second quadrouple, they enter a field free collision chamber. What gas is pumped through?
Helium
What does the term field free mean?
The quadrouple is operated in radio-frequency-only-mode and no dc potential is applied across the rods
This mode provides and excellent method of focussing the ……… ….. but does not act as an ….. …….?
Scattered ions, ion filter
The collisions of the fast-moving parent ions and argon atoms cause what?
Further fragmentation of parent ions or precursor ions
What does this give?
daughter ions
What happens in the third quadrouple
The spectrum of daughter ions is then scanned
For quantitative analysis of target compounds, what mode is the third MS operated in?
the selected-reaction monitoring mode
What is selected reaction monitoring?
a non-scanning MS technique performed on tandem mass spectrometers
Collision-induced dissociation is used as a means to increase what?
selectivity
In SRM, what are the 2 mass analyzers used as?
static mass filters
What are these used to monitor?
a particular fragment ion of a selected precursor ion
What are the specific pair of m/z values associated to the precursor and fragment (daughter) ions selected referred to as?
a transition and can be written as a parent m/z > fragment m/z
In SRM analysis, there is generally no mass spectra recorded. Instead the detector acts as a counting device for what?
ions matching selected transition thereby returning an intensity value over time
How can multiple reaction monitoring and selective reaction monitoring be measured at the same time on the chromatographic time scale?
by rapidly toggling between the different precursor/fragment (daughter) pairs
How does the method allow for additional selectivity?
By monitoring the chromatographic coelution of multiple transitions for a given analyte
