MASS SPECTROMETER

Analytical Instrumentation

4/28/15

Mass Spectrometer

Introduction
It is mainly used in process industries
Multi component analysis
For closed loop control with high accuracy, reliability
and determinism

4/28/15

Mass Spectrometer

Principle
They separate the ion of interest from the sample by
their mass to charge ratio
It is a simple vacuum device which creates, separates
and detects an electron by reducing the collision
between the molecules and ions, which is done by
increasing the mean free path of the molecule
For which a vacuum of order 10-2 to 10-10 is required
4/28/15

Mass Spectrometer

Essential Parts
A vacuum envelope
Inlet leak
Evacuation pump

4/28/15

Mass Spectrometer

4/28/15

Mass Spectrometer

Schematic of mass
spectrometer

4/28/15

Mass Spectrometer

Operation
In a typical MS procedure, a sample, which may be solid, liquid, or gas, is
ionized, for example by bombarding it with electrons. This may cause some
of the sample's molecules to break into charged fragments.
These ions are then separated according to their mass-to-charge ratio,
typically by accelerating them and subjecting them to an electric or
magnetic field: ions of the same mass-to-charge ratio will undergo the same
amount of deflection.
The ions are detected by a mechanism capable of detecting charged
particles, such as an electron multiplier. Results are displayed as spectra of
the relative abundance of detected ions as a function of the mass-to-charge
ratio.
The atoms or molecules in the sample can be identified by correlating known
masses to the identified masses or through a characteristic fragmentation
pattern.
4/28/15

Mass Spectrometer

Hard ionization and soft

ionization
Hard ionization techniques are processes which impart high quantities of
residual energy in the subject molecule invoking large degrees of
fragmentation (i.e. the systematic rupturing of bonds acts to remove the
excess energy, restoring stability to the resulting ion). Resultant ions tend to
havem/zlower than the molecular mass (other than in the case of proton
transfer and not including isotope peaks). The most common example of
hard ionization is electron ionization (EI). (GMC is used)
Soft ionization refers to the processes which impart little residual energy
onto the subject molecule and as such result in little fragmentation.
Examples includefast atom bombardment(FAB),chemical
ionization(CI),atmospheric-pressure chemical
ionization(APCI),electrospray ionization(ESI),matrix-assisted laser
desorption/ionization(MALDI). (HPLC are used)
4/28/15

Mass Spectrometer

Types of ionisation
Atmospheric Pressure Chemical Ionisation (APCI)
Chemical Ionisation (CI)
Electron Impact (EI)
Electrospray Ionisation (ESI)- biochemical analysis
Fast Atom Bombardment (FAB)
Field Desorption / Field Ionisation (FD/FI)
Matrix Assisted Laser Desorption Ionisation
(MALDI)
Thermospray Ionisation (TSP)
4/28/15

Mass Spectrometer

Electrospray ionisation

4/28/15

Mass Spectrometer

10

Electrospray ionisation
Well-suited to the analysis of polar molecules ranging from less
than 100 Da to more than 1,000,000 Da in molecular mass.
the sample is dissolved in a polar, volatile solvent and pumped
through a narrow,stainless steel capillary (75 - 150
micrometers i.d.) at a flow rate of between 1 uL/min and 1 mL/min
Ahigh voltageof 3 or 4 kV is applied to the tip of the capillary,
which is situated within the ionisation source of the mass
spectrometer, and as a consequence of this strong electric field,
the sample emerging from the tip is dispersed into anaerosol of
highly charged droplets, a process that is aided by a co-axially
introducednebulising gasflowing around the outside of the
capillary
4/28/15

Mass Spectrometer

11

Electrospray ionisation
Ahigh voltageof 3 or 4 kV is applied to the tip of the
capillary, which is situated within the ionisation source
of the mass spectrometer, and as a consequence of this
strong electric field, the sample emerging from the tip is
dispersed into anaerosol of highly charged
droplets, a process that is aided by a co-axially
introducednebulising gasflowing around the outside
of the capillary
This gas, usually nitrogen, helps to direct the spray
emerging from the capillary tip towards the mass
4/28/15
Mass Spectrometer
spectrometer

12

Electrospray ionisation
The charged droplets diminish in size bysolvent
evaporation, assisted by a warm flow of nitrogen
known as thedrying gaswhich passes across the front
of the ionisation source.
Eventually chargedsample ions, free from solvent, are
released from the droplets, some of which pass through
asampling coneor orifice into anintermediate
vacuum region, and from there through a small
aperture into the analyser of the mass spectrometer,
which is held underhigh vacuum. The lens voltages
4/28/15
Mass Spectrometer
13
are optimised individually
for each sample

Matrix assisted laser desorption

ionisation

4/28/15

Mass Spectrometer

14

Matrix assisted laser desorption

ionisation
Deals well with thermolabile, non-volatile organic compounds
especially those of high molecular mass and is used successfully in
biochemical areas for the analysis ofproteins, peptides,
glycoproteins, oligosaccharides, and oligonucleotides.
It is relatively straightforward to use and reasonably tolerant to
buffers and other additives.
The mass accuracy depends on the type and performance of the
analyser of the mass spectrometer, but most modern instruments
should be capable of measuring masses to within 0.01% of the
molecular mass of the sample, at least up to ca. 40,000 Da.
4/28/15

Mass Spectrometer

15

Matrix assisted laser desorption

ionisation
MALDI is based on thebombardmentof sample molecules
with alaserlight to bring aboutsample ionisation.
The sample is pre-mixed with a highly
absorbingmatrixcompound for the most consistent and
reliable results, and a low concentration of sample to matrix
works best.
The matrix transforms the laser energy intoexcitation
energyfor the sample, which leads to sputtering of analyte
and matrix ions from the surface of the mixture.
4/28/15

Mass Spectrometer

16

Matrix assisted laser desorption

ionisation
In this way energy transfer is efficient and also the
analyte molecules are spared excessive direct energy
that may otherwise cause decomposition.
Most commercially available MALDI mass spectrometers
now have a pulsed nitrogen laser of wavelength 337
nm.

4/28/15

Mass Spectrometer

17

Matrix assisted laser desorption

ionisation
The sample to be analysed is dissolved in an appropriate
volatile solvent, usually with a trace of trifluoroacetic acid if
positive ionisation is being used, at a concentration of
ca.10 pmol/uLand an aliquot (1-2 uL) of this removed
and mixed with an equal volume of a solution containing a
vast excess of a matrix.
A range of compounds is suitable for use as
matrices:sinapinic acidis a common one forprotein
analysis whilealpha-cyano-4-hydroxycinnamic acidis
often used forpeptideanalysis.
4/28/15

Mass Spectrometer

18

Matrix assisted laser desorption

ionisation
An aliquot (1-2 uL) of the final solution is applied to the sample target which
is allowed to dry prior to insertion into the high vacuum of the mass
spectrometer.
The laser is fired, the energy arriving at the sample/matrix surface optimised,
and data accumulated until a m/z spectrum of reasonable intensity has been
amassed.
The time-of-flight analyser separates ions according to theirmass(m)-tocharge(z) (m/z)ratios by measuring the time it takes for ions to travel
through a field free region known as the flight, or drift, tube.
The heavier ions are slower than the lighter ones.
4/28/15

Mass Spectrometer

19

Matrix assisted laser desorption

ionisation
The m/z scale of the mass spectrometer iscalibratedwith a
known sample that can either be analysed independently
(external calibration) or pre-mixed with the sample and matrix
(internal calibration).
MALDI is also a "soft" ionisation method and so results
predominantly in the generation ofsingly charged
molecular-related ionsregardless of the molecular mass,
hence the spectra are relatively easy to interpret.
Fragmentation of the sample ions does not usually occur.
4/28/15

Mass Spectrometer

20

Matrix assisted laser desorption

ionisation
Inpositive ionisationmode theprotonated molecular ions
(M+H+)are usually the dominant species
Positive ionisation is used in general
forproteinandpeptideanalyses.
Innegative ionisationmode thedeprotonated molecular
ions
(M-H-)are usually the most abundant species
Negative ionisation can be used for the analysis of
oligonucleotidesandoligosaccharides
4/28/15

Mass Spectrometer

21

Types of analysers
Magnetic Sector
Time of flight
Quadrupole filter
Ion trap instruments (3D Quadrupole arrangement)

4/28/15

Mass Spectrometer

22

Magnetic Sector
Two types of magnetic sector: fixed permanent magnet,
electromagnetic sector
Right angled field creates a bending beam
proportional to the m/z ratio
Scanning by multiple collectors fixed at predetermined
focal point
4/28/15
Control is achieved by voltage
or direct field control
Mass Spectrometer

23

4/28/15

Mass Spectrometer

24

Time of flight
Subjected to ve polarity field and passed through a
drift region
The lighter ions travels faster than the lighter ones
T=k(M/E)0.5
Discrete pulsed sampling which samples at and around
20-30k pps
Samples enter separated based on mass numbers
ionized detected at cathode
using photomultipliers
4/28/15
Mass Spectrometer

25

4/28/15

Mass Spectrometer

26

Quadru-pole filter
Four cylindrical rods orthogonally
Ion undergoes complex traverse motion
Counter polarity arrangement that opposite poles are
evenly charged
Both are 180 degrees out of phase with RF and DC
intermixed with DC similar to the polarity of coil
By altering the voltages of the rods passband is varied
which is also its unique characteristics aiding in its high
resolution
Pass band allows only a specific mass to flow through
4/28/15
Mass Spectrometer
27
whereas other are neutralised
(happens in both the

4/28/15

Mass Spectrometer

28

Tandem mass spectrometry

Used to producestructural informationabout a
compound by fragmenting specific sample ions inside
the mass spectrometer and identifying the resulting
fragment ions
Also enables specific compounds to be detected in
complex mixtures on account of their specific and
characteristic fragmentation patterns

4/28/15

Mass Spectrometer

29

Tandem mass spectrometry

Has more than one analyser, in practice usually two,
separated by a collision cell into which an inert gas (e.g.
argon, xenon) is admitted to collide with the selected
sample ions and bring about their fragmentation,

4/28/15

Quadru-pole quad-rupole
magnetic sector quadru-pole
magnetic sector magnetic sector
Quadru-pole time-of-flight

Mass Spectrometer

30

Questions for further reading

Why in hard ionization techniques HPLC cannot be used??
Application areas of MS techniques??
What will happen when MS is applied to analyse different molecules that
posses geometrically and optically same isomeric characteristics??
What will happen when MS is applied to analyse different molecules that
posses similar fragmented ions??
How these MS techniques can be incorporated along with chromatographic
analysis??
4/28/15

Mass Spectrometer

31

References
http://
www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm
Instrument engineers Handbook B.G.Liptak
Process measurement and instrumentation Library MIT
online resources
A survey on high resolution mass spectroscopic
analysers springer-vale publications

4/28/15

Mass Spectrometer

32

THANK YOU

4/28/15

Mass Spectrometer

33