6. Mass Spectrometry
Adv. Inst. Techs
How does it work?
• a very small amount of sample is bombarded by a beam of high energy
(usually electron beam)
• produces a positively charged species
•
M  M+ + e
• known as the molecular ion (same mass as the parent compound)
• target molecules fly apart into a number of smaller pieces
• all the positive ions are separated by masses to produce the spectrum
• peaks corresponding to the break up of the molecule are called the
fragmentation pattern
base peak
fragmentation
pattern
135
77
intensity (%)
104
165
180
molecular ion
m/e (mass to charge ratio)
Interpreting mass spectra
1. Molecular weight
• in most cases, some of the molecular ion remains unfragmented
• it produces a peak in the spectrum which is the highest mass peak
• this mass peak is most likely equal to the molecular weight of the
compound
• known as the molecular ion
• example spectrum - 180
• most intense peaks will have a peak 1 higher (due to presence of C-13)
2. Molecular fingerprint
•
•
•
•
each molecule will always fragment in the same way
compounds may be positively identified by a comparison of their mass spectra
MS of very similar compounds are very similar
therefore, not as good a “fingerprint” as IR
3. Isotope peaks
• MS shows a peak for each isotope of a mixture
• see the peaks due to both the 37Cl and 35Cl and these always occur in the natural
3:1 ratio
• bromine shows peaks for 79Br and 81Br in 1:1 ratio
4. The “nitrogen rule”
• a molecule of even number MW must contain either no nitrogen or an even
number (or no) of N’s
• an odd MW signifies an odd number of Ns
• it doesn’t matter what other elements are in the compound
5. Characteristic fragmentation losses
• some functional groups fragment in a particular and consistent way
• eg ethyl esters always lose the OCH2CH3 group
• show a strong peak 45 less than the molecular ion
Exercise 6.1
What loss would you expect to see from the molecular ion if the compound was a methyl ester?
• 31 (CH3O)
105 <= 150
99 <= 144
Exercise 6.2(a)
COOCH2CH3
loss of 45 =>
possibly ethyl ester
FW 165
NH2
odd FW:
odd no. Ns
Exercise 6.3(b)
COOCH3
even FW:
0 or even
no. Ns
Presence of Br
FW 214 & 216
Br
Instrumental components
Sample
Inlet
Fragmentation
Chamber
Mass
Analyser
Detector
high
vacuum
Sample inlet
• a very small quantity of sample (10-100 g) is required in the mass
spectrometer
• also necessary that the sample is carried into the high vacuum within the
instrument without leaks
• for substances with relatively high volatility, the sample is introduced into a
heated injection port at 300C, very much like one in a GC
• the sample inlet can be a connection to the “end” of a GC column
• if the material is not volatile or is unstable when heated, then a direct
injection method is used, where the sample is held in a tiny cup, and
inserted directly into the fragmentation chamber
Ion source
• MS relies on the detection of charged fragments of the original molecules
• the analyte must be ionised first
• a number of methods for achieving this, including:
• electron impact – the most common; the only one you need to know
• chemical ionisation
• fast atom bombardment
Electron impact ionisation
• a beam of high energy electrons fired across the path of the analyte
molecules
• an electron is removed by the beam, and energy is added
• M + e → M+* → F1+, F2+, F3, F4• only the positive (the most prevalent) fragments are analysed
• the energy of the beam is controlled by the applied voltage (70 V is normal);
lower voltage means less energy means less fragmentation
Caffeine fragmentation
Vacuum
• is very substantial: around 10-7 torr (1 atm is 760 torr)
• requires two levels of vacuum pump: one to remove the bulk of the air, and
the ultra high vacuum pump to remove the last traces
Exercise 6.3
• Why is such a high vacuum required?
• to remove contaminants (eg O2, N2)
• to increase the stability of the fragments by reducing chance of collision
Mass analyser
• the equivalent of a monochromator
• separates the fragments on the basis of their different masses
• before the mass analyser is an ion filter, which blocks any non-positive
fragment from passing into the mass analyser
• employs a series of negatively charged plates, with increasing voltages
-
F+
FF
--
---
Magnetic sector mass analyser
• particles moving through a magnetic field have their path bend at a radius
proportional to their mass: the heavier the mass, the wider the curve of its
path
• the magnetic field is changed during the scan to allow different masses
through
particle
with
wrong
mass for
curve
magnetic
field
curved
path
particle
with
correct
mass for
curve
Quadrupole mass analyser
• answers to Exercise 6.4
• four metals poles aligned to create electrical fields that allow one mass
through at a given setting
Comparison
• the magnetic sector (MS) is physically larger than the quadrupole (Q)
because of the large magnet
• the MS cannot scan as quickly as the Q
• the MS costs more than the Q
• the MS is capable of much better resolution (0.001 amu) than the Q (0.1
amu)
• Q is ideal as a detector for another instrument eg GC, HPLC, ICP (known as
hyphenated instruments)
• MS is better for freestanding MS instruments
GC-MS
• the MS needs very small quantities, which equates to the amounts of
analytes coming from capillary GC columns
• the carrier gas is a problem because it would swamp the MS
• separated and removed by differences in momentum for light He
MS inlet
GC
column
vacuum
pump
How does the MS produce a chromatogram?
• at least once a second, the MS does a full mass scan
• the electronics generate two measures from the detector:
• the mass spectrum which is stored
• the total number of fragments (of all masses) in the scan – this is plotted
on the chromatogram
• when only carrier gas is eluting, few fragments are generated
• when a compound elutes, more fragments are generated, giving a peak
• the chromatogram then looks the same as if a TC or FI detector had been
used
The great advantage of a MSD
• each point in the chromatogram is linked to a MS
• this can be used to identify the compound (in conjunction with a spectral
library)
• no need for spiking or RT comparison
Making the MSD selective
• setting the mass analyser to a fixed mass which is characteristic of the
analyte (equivalent to quantitative analysis on UV-VIS)
• known as single (or selected) ion monitoring
• no scanning involved (no ID possible, but not necessary as you know what
you are looking for)
• peaks only generated by compounds producing a fragment of the specified
mass
• simplifies chromatogram
• increases sensitivity (all fragments of that mass are detected – in TIC, most
are lost)
Comparison with other GC detectors
• MS is as sensitive as FI
• MS is much more expensive than other detectors
• no other detectors can identify the peak
HPLC-MS
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•
•
•
•
•
the problem of removing the mobile phase is much harder with a liquid
must be instantaneous and must not “damage” analytes
solution was electrospray
uses a high voltage electrical field applied to a nebuliser
this vapourises the solvent and ionises the analytes at the same time
HPLC-MS is the standard “drugs-in-sport” testing method
ICP-MS
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•
•
•
•
•
•
•
same front end: plasma torch
not used to produce characteristic radiation, but a stream of ions
obviously different to GC- & HPLC-MS which analyse organic molecules
this analyses elements
no fragmentation occurs (these are atoms already!!)
very expensive
far more sensitive than any commercially available instrument (ng/L)
able to distinguish between isotopes of same element