mass spectrometry in mcalhome.cc.umanitoba.ca/~wardat/ms lecture 2017.pdf · mass spectrometry in...
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Mass Spectrometry in MCAL
• Two systems: GC-MS, LC-MS
– GC seperates small, volatile, non-polar material
– MS is detection devise (Agilent 320-MS TQ Mass Spectrometer)
• Full scan monitoring
• SIM single ion monitoring
• MSMS monitoring
• Sample can be injected as a liquid, a gas or even a solid
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Different Types of MS
• GC-MS - Gas Chromatography MS– separates volatile compounds in gas column and ID’s by mass
• LC-MS - Liquid Chromatography MS– separates delicate compounds in HPLC column and ID’s by
mass
• MS-MS - Tandem Mass Spectrometry– separates compound fragments
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How Does MS Measure Mass
• Deflection of ions in electric or magnetic
field which is related to mass of ion.
• Ions need to be charged. Can be either
positive or negative.
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GC-MS
Injection port
ProbeGC 3800
MS 320
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GC-MS
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EI Fragmentation of CH3OH
CH3OH CH3OH+
CH3OH CH2O=H+ + H
CH3OH + CH3 + OH
CHO=H+ + HCH2O=H+
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EI Breaks up Molecules in Predictable Ways
Molecular ion
Electron Impact MS of CH3OH
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The GC-MS
=>
A mixture of compounds is separated
by gas chromatography, then identified
by mass spectrometry.
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Liquid sampling syringe
Heating block with stirrer
Headspace vials
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Probe (solid
samples)
Sample
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Probe
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Triple Quodrapole
CID in
this
quod
ms-ms
Collect full scan or
SIM
Collect ms-ms
fragment ions in
this quodDetector
Ion
volume
From GC
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Mass Spectrometer (Q3)
Detector
photomultiplier
Diffusion pump
Q1
Q2
Q3
Sample from GC
Ion Source tungsten filament
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GC-MS
• The MS is equipped with a triple
quadrupole analyzer and allows several
types of mass detection to be performed.
• Full scan (direct MS) mode is used.
• Sim can be used
• MS-MS can be used
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Key Components of MS
• Ionization – gas phase ions created in source
• High vacum – creates free path
• Mass Analyser- sorts ions by M/Z ratio
- electric field
• Detector – creates signal multiplication
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GC Settings
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Varian (Agilent) FactorFour
Capillary GC Columns.
• Capillary column: thin fused-
silica capillary.
• 50 m in length and 250 µm
inner diameter.
• The stationary phase is CP-
sil 8, with 5% phenyl 95%
dimethyl polysiloxane
coated on the inner surface.
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Electron Ionization
• It uses a heated filament to produce
electrons. The filament is usually made of
rhenium or tungsten.
• Once the electrons are produced they are
accelerated through a potential difference
of around 70V this gives electrons with
70eV of energy.
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Electron Impact
• Electrons produced by the source will then collide with the sample and remove an electron to give ions
e- + M M+. + 2e-
• Fragmentation is a result of an excited molecular ion, which in attempting to gain stability
• Fragmentation is not random and occurs through real chemical reactions.
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Caffeine
fragmentation
pattern GC-MS 70eV
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Caffeine Fragmentation Pattern
109
194
137 165
55
67
82
44
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Q1 Window
Ion Range
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Various constituents
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Quantitation of Ingredients
• You usually quantitate the ingredient that
you are interested in by:
– SIM which measures one of the ions in your
sample
or
– MSMS which measures one of the ions
produced in the collission cell (Quad 2)
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Mass 294
Q1 only
Single ion monitoring SIM or SIR
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Fractionating the 294 ion in Quad 2 will produce product ions which
can be scanned at 100 to 300 to identify the product ions.
MW 294 -
molecular ion
Product ions
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MSMS product ions 183-185
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Mass Spectrometry in MCAL
• Two systems: GC-MS, LC-MS
– LC seperates small, volatile, polar and non-
polar material
– MS is detection devise (Agilent 500-MS
IonTrap (IT) Mass Spectrometer
• Full scan monitoring
• SIM single ion monitoring
• MSMS monitoring
• Liquid samples are analysed
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Agilent LCMS
• The LCMS can be used either with the LC
seperating the sample on the HPLC
column before introduction into MS
or
• Stand alone MS where sample is
introduced via the syringe pump.
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LC-MS
ES
Pumps
Injection valve
Syringe
pump
Column
mixer
Sample rack
(autosampler)
solvents
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From HPLC or Syringe
pump
Source
N2 drying gas @150-
300OC Scimmer cone
Hexapole
Ion guide
ES unit
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Electrospray Ionization
• During standard electrospray ionisation the sample is dissolved in a polar, volatile solvent and pumped through a narrow, stainless steel capillary (needle).
• A high voltage of 3 to 6 kV is applied to the tip of the needle, which is situated within the ionisation source of the mass spectrometer
• As a consequence of this strong electric field, the sample emerging from the tip is dispersed into an aerosol of highly charged droplets,
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Electrospray Ionization
• The charged droplets diminish in size by solvent evaporation, (N2 drying gas)
• Charged sample ions, free from solvent, are released from the droplets,
• Some pass through an orifice in the cone into an intermediate vacuum region,
• Then into the analyser of the mass spectrometer, which is held under high vacuum.
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Electrospray Ionization
Spray Needle Cone
Atmospheric Pressure
To MSCapillary
N2
flow
Liquid from
Syringe pump
or LC
Solvent
droplets
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Electrospray Ionization (ESI)
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500-MS Operation
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500 –MS Operation
• Gas phase ions are generated from
solution at atmospheric pressure using
either Electrospray Ionization (ESI) or
Atmospheric Pressure Chemical Ionization
(APCI).
• The ions are transported through two
vacuum interfaces via a metal capillary
tube.
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500-MS Operation Continued
• After exiting the capillary, the ion stream expands into a supersonic beam.
• The sample passes through a skimmer cone (which is at ground potential). If the potential difference between the capillary and skimmer cone is high enough, some molecules may fragment (Capillary Induced Dissociation (CID)).
• After passing through the skimmer cone, ions enter an 8 m Torr vacuum chamber containing ion optics.
• The ions enter a hexapole ion guide. The ion guide uses an oscillating potential across six cylindrical rods to confine the ions and a direct current (dc) potential to facilitate ion transport. Collisions with neutral gas in the ion guide reduce the kinetic energy of the ions to facilitate focusing.
• The ions pass through the ion guide exit and focus lenses,
• The ions then pass through a split cylinder that acts like a gate for the ion trap by either focusing the beam or deflecting it. The final end cap lens focuses the ions through the entrance hole of the trap.
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Electron Multiplier
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Ion Trap
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Operation of Ion Trap
• There is a hole in the center of both the entrance and exit end cap electrodes. This hole allows the ions to enter the trap and exit to the detector. The holes in the edge of the end caps contain posts that make contact with pogo pins that carry supplemental waveform signals.
• An RF generator provides a high voltage, 781 kHz RF, that is applied to the ring electrode. With the proper RF voltage, the ion trap electrodes create a three-dimensional, hyperbolic electric field.
• This field traps the ions in stable orbits. In the presence of helium damping gas, the ions are cooled towards the center of the trap.
• As the RF voltage increases, the ion trajectories become unstable in increasing order of mass to charge ratio. The ion trap ejects the ions and sends them to the conversion dynode and then to the electron multiplier for detection.
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500-MS Detector
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Ion DetectionAfter exiting the trap, ions accelerate toward an off axis conversion dynode that generates a combination of positive ions and electrons through secondary electron emission.
For the detection of positive ions, the conversion dynode is set to a large negative voltage (typically -15 kV). The secondary electrons are attracted to the relatively positive multiplier.
Electrons or ions emitted from the conversion dynode strike the cathode with sufficient velocity to dislodge additional electrons from the inner curving surface of the cathode. The increasingly positive potential gradient draws the ejected electrons into the electron multiplier, further accelerating them in the process., the ejected electrons strike the curved inner surface of the multiplier, resulting in the emission of more electrons.
This configuration produces a cascade of electrons that accelerate toward ground potential at the exit end of the cathode.
The anode collects the electrons and passes the resulting ion signal to the ion amplifier. The ion current is proportional to the total number of ions the ion trap ejects.
Each electron or positive ion that enters the electron multiplier generates approximately 10^5 electrons.
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Separation Of Analgesics
• Seperation is based on HPLC and mixture
of Tylenol (acetaminophen, caffeine) and
aspirin are separated.
• MS substance in mixture, can be
optimized for each of RF, needle voltage,
capillary voltage and CID.
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Seperation Under Optimum
Conditions• Precursor (parent) ion produces
breakdown ions (daughter ions) which can
be used to quantitate the substances in a
HPLC separated mixture.
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Caffeine
fragmentation
pattern