Mass Spectroscopy
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Transcript Mass Spectroscopy
Mass Spectrometry
Dr. Peter Sharp
Brighton and Sussex University
Hospital Trust
Principles of Mass Spectrometry
Mass Spectrometers have three basic
functions
1 Vaporisation
2 Ionisation
3 Ion separation according to mass /charge
m/z
Since z usually equals unity A mass
spectrometer produces and weighs ions
Ionisation - the only obstacle to a
successful analysis.
First experiments 1930s Chapman
late 1960s Dole developed practical method
1980s Fenn and Mann modern techniqueion spray- multiple charging of
macromolecules-important development for
the analysis of post-translational proteins
following the human genome project.
Electron impact
ionisation/fragmentography
This is where gaseous effluent from usually a GC
is bombarded with electrons from a filament
Traditionally 70 eV used for ionisation and when
most chemical bonds have 4- 7 eV of energy it is
not surprising to see extensive fragmentation
producing both +ve and –ve ions.
NBS and Pfleger libraries of fragmentation
patterns.
Disadvantage – few Molecular ion species remain
Chemical ionisation
This is where a secondary gas or nebulised
component is ionised first then passes that
energy of ionisation onto the target species.
In this way the ionisation is softer than the
70 eV and considerably more molecular ions
are seen
Reagent gases include methane, ammonia
and isobutene for positive ion ionisation.
Heated Nebulizer - APCI
Curtain Gas
.
Make-up Air
Heat
Liquid
H O
2
M
Air
for
Nebulization
Heat
+
H3O M
M+H
250u orifice
Curtain Gas
Ionisation from solids
MALDI – Matrix assisted Laser Desorption
ionisation
Used for larger molecules such as peptides
and proteins
API Analytical Domains
Ionic
Analyte Polarity
IonSpray
Heated
Nebulizer-APCI
GC/MS
Neutral
101
102
103
Molecular Weight
104
105
Types of ion separation
1 using static radio frequency/magnetic
fields only and the path and/or velocity eg
sector instruments and time of flight.
2 Using Dynamic fields of Radio frequency
with oscillating electrical fields which
selectively stabilises and destabilises the
ions eg ion trap and quadrupole instruments
Definitions
Radio-frequency: A frequency or range of
oscillation between 3 Hz and 30 GHz
corresponding to frequency of alternating
current electrical signals used to produce
and detect radio waves. These have
electro-magnetic forces
Schematic sector mass spec
More modern instruments
Quadrupole
Ion Trap
Time of Flight
Fourier Transform Ion Cyclotron Resonance
Tandem Mass spectrometers which may be
any combination of two of them
Quadrupole
Uses oscillating electrical fields to stabilise
and destabilise ions passing through a RF
quadrupole field.
Quadrupole TMS uses two analytical
quadrupole analysers separated by a
collision cell
Usually in mass range to 1000 daltons
Not very high resolution 0.5 Dalton
Ion Path of TMS
Ion Trap
Ion trap and quad
Ion trap
Ions pulsed into trap with oscillating repeller
electrode –Vopen/+Vclosed
Ions trapped and orbit in RF field set by annular
electrode
Ions ejected from trap by increasing RF or adding
DC current via end cap electrodes
Advantage is trapping and holding large numbers
of ions increasing signal:noise ratio.
Disadvantage: Space-charge effect where too
many ions distort the electrical field and impair
performance
Time of Flight
Accelerates ions using an electric field
through the same potential and measures
the time to the detector.
Need a relatively long flight path and
therefore can be large instruments.
Smaller ions go faster
Can measure mass to 4 decimal places!
Therefore good for isotope identification
Fourier Transform ion cyclotron
resonance
Measures mass by detection of the image current of ions
cyclotroning in a magnetic field
The ions are injected into a Penning Trap(a static
electric/magnetic ion trap) where they effectively form part
of a circuit. Detectors at fixed positions measure the
electrical signal(weak AC ) when the ions pass near them.
Since the frequency of cycling is a function of
mass/charge, this can be deconvoluted by performing a
Fourier transform on the signal.
Since a single ion is counted more than once this
technique has high sensitivity, high resolution and
precision.
Ion cyclotron resonance
Ions detected without colliding with detector via
the image current
Ions can be excited by pulses of RF which boosts
the image current which then fades back to the
unexcited state
Through Fourier transformation this fade in current
yields increased information on the mass
spectrum
Good for accurate mass measurement of proteins
MS/MS: Modes of Operation
Product Ion Scan
Select Precursor
Ion
CAD
Scan Products
Precursor Ion
Scan
ScanPrecursor
Ion
CAD
Select Product Ion
Constant
Neutral Loss
Scan precursor and product ions at a constant mass off-set between Q1 and Q3
Type of MS/MS Scans
Product Ion Scan
m1+ set
Product ion spectrum of a particular compound
m2+
m2+
+
m2 scan
m1+
m2+
Precursor Ion Scan
m1+ scan
A set of compounds with a common product ion
m2+
m1+
m1+
m1+
m2+ set
Constant Neutral Loss Scan
m1+ scan
A set of compounds with a common neutral fragment
+
m
m2+ scan
m2+
m2+
m1
-m
-m
m1+
Multiple reaction monitoring
Morphine
codeine
dihydrocodeine
6-acetyl morphine
methadone
dipipanone
moramide
pentazocine
propoxyphene
paracetamol
amphetamine
286 -152
300 -152
302 -199
328 -165
310 -265
350 - 265
393 - 306
286 - 218
340 - 58
152 - 110
136 - 91
Mdma
cocaine
benzoylecgonine
chlormethiazole
tramadol
phenethylamine
methaqualone
nalbuphine
buprenorphine
noscapine
hydroxyzine
194 - 163
304 - 182
290 -168
162 - 113
264 - 58
122 - 77
251 - 132
358 - 161
468 - 414
413 - 220
375 - 201
The molecular ion species of syringe
contents
Peter Sharp:
heroin
noscapine
Acetyl codeine
papaverine
paracetamol
Acetyl morphine
cocaine
Problems with mass spectrometry
Ionisation suppression – some molecules ionise
much easier than others and those with a greater
avidity for protonation may cause suppression of
ionisation of those less polar compounds.
Use standard addition quantitation techniques to
minimise effects of ionisation suppression
Specificity of ions – nortriptyline, norvenlafaxine
and Tramadol all have same MW 263 and all can
produce the same daughter ion mass 58
More drawbacks
Too many ions cause interference –
especially in ion traps – like all forms of
separation/chromatography any system can
be under/overloaded. Good results
obtained with optimal ion concentrations
Isotope c-12, c-13 and Cl-35 and Cl-37
produce reduced specificity with machines
of lower resolution eg unity MW resolution
Low MW daughter ions have less specificity