MASS SPECTROMETRY(mass-spec) -2013 - P.ravisankar- WHAT ABOUT MASS SPECTROMETRY,BASIC PRINCIPLE,INSTRUMENTATION, ION SOURCES, MASS ANALYZERS,APPLICATIONS.

Mass spectrometry(Mass spec)
Prof.P.Ravisankar
Vignan Pharmacy college
Valdlamudi
Guntur Dist.
Andhra Pradesh
India

Still spectroscopy?
• the interaction of electric and/or magnetic fields (i.e.
eletromagnetic radiation) with matter to determine weight
or mass.
• Measures mass, not absorption or emission of
electromagnetic radiation

The concept of mass spectrometry was first put forth by
Sir J.J Thomson, English Physicist Who discovered the electron in 1887.
He got 1906 Nobel Laureate in Physics.
We have to first ionize and impart the charged on those particles ..
In order to respond electric field and magnetic field..
Quickly remind you Thomson-II experiment a beam of electrons
Just use magnetic field close to the beam
because the beam is charged .. Its just gets deflected ..
How much the beam(that Particles are) is deflected depends on how heavy they are and
Their charge is..
We use the exact principle,(same basic technique )in Mass-spec.

Cathode rays are stream of –ve charges
Cathode rays bend towords +ve charged plate
Cathode rays bend near magnets.

Mass spectrometry is essentially a technique for "weighing" molecules.* Obviously, this
is not done with a conventional balance or scale. Instead, mass spectrometry is based upon
the motion of a charged particle, called an ion, in an electric or magnetic field. The mass
to charge ratio (m/z)** of the ion effects this motion. Since the charge of an electron is
known, the mass to charge ratio a measurement of an ion's mass.
The concept of MS is to form ions from a sample,
separate the ions based on their mass-to-charge ratio (this can be
considered to be the same as the mass because the ion has only
a single charge in most cases), and measure the abundance of
the ions.

History:
In the early 1900’s while working on electromagnetic radiations, the ions generated
By the gases in the cathode ray tube led to the discovery of Mass spectrometer.
Francis William Aston a physicist working in Cambridge England 1919 worked on
Mass spectrometry and established technique for the measurement of atomic mass.
Francis William Aston, English physicist,
student of Thomson, and He was awarded
1922 Nobel prize in Chemistry.
1948-52 - Time of Flight (TOF) mass analyzers introduced
1955 - Quadruple ion filters introduced by W. Paul, also invents the ion trap in 1983 (wins
1989 Nobel Prize)
1968 - Tandem mass spectrometer appears
Mass spectrometers are now one of the MOST POWERFUL ANALYTIC TOOLS IN CHEMISTRY.

What does a mass spectrometer do?
Mass –spec or simply MS is a super important technique
Mass spec is easy technique to give you Molecular weight(from molecular ion (M+)
You can get Molecular formula (we talk elements about by HRMS)
and some of the fundamental things.
One thing that Mass spec can easily,easily,easily talk to youabout elements presen
Nearly in the periodic table can be determined by mass spectrometry.
MS is really important technique because that You can easily see Br,Cl and you
Can see S and Si you are looking for..
NMR is not going to be a technique that talks to you about elements
like that and IR is not going to be a technique that talks to you …
This is why we should study about Mass spec..
MS is incredible valuable in getting structure (from fragments) .
Infact Bio molecular MS to sequencing peptides and proteins and also
natural products and also organic structures.
Structures (from fragmentation proces)Hard ionization
(slash techniques) MSMS( taking ions and
deliberately dashing in to them smashing them and see how they look like.
It measures mass better than any other technique.
It can give information about chemical structures.

MS was originally used to determine the existence of the stable isotopes of the
elements in the periodic table..1/ [The word isotope was suggested by Frederick Soddy
(1877–1956) as a student and collaborator of Ernest Rutherford at McGill
University in Montreal, Canada, in 1913. Isotopes are different forms of the same
element that have the same atomic number, but differ in their relative atomic
mass due to a difference in the number of neutrons present in the
nucleus of the atom. The word is derived from the Greek words isos (equal) and
topos (places), ‗having the same place‘ in the periodic table.
MS plays an important role not only in organic and biochemistry but also in inorganic
chemistry such as the determination of metal contaminants in silicone
wafers(silicone crystal,in electronics), drinking water, soils, industrial
waste, etc. To quantitate unambiguously identified sub picogram amounts of
the pesticide Malathion on orange peel, age of artefacts [isotope-ratio
mass spectrometry (IR MS)], contamination of the surface of metal and composite
It is used to determine the airplane wings [secondary
ion mass spectrometry (SIMS)], and the components that give fresh-baked bread its
delightful aroma.
One of most powerful analytical tools MS is sensitive (10-6 to <10-13 g)
But Complex instrumentation, expensive,structure obtained indirectly
• complex spectra/fragmentation for hard ionization sources
• simple spectra for soft ionization sourcesThe list of applications are endless.

It is also used for the identification and quantitation of various organic substances from the
simplest gases such as methane and halomethanes to complex biomolecules
such as proteins, oligonucleotides, and noncovalent complexes.
Mass spectrometers are not only found in analytical
laboratories but also inside the helmets of space suits (to
determine the level of gases that may pose a hazard),
in tanks and other battlefield vehicles and on ships and
aircraft (to detect the presence of chemical and biological
warfare agents), and as more conventional field-portable
instrumentation for use at crime scenes, hazardous.
SIMS technique was used to study the impurities in material
such as the aluminium used for airplane wings and germanium
wafers used in early
solid-state electrical devices.
SIMS and its variants are widely used in the analysis and study of
surfaces of all
types of material – from papers used in laser printers, to
microprocessor devices, to the wings of aircraft manufactured from
many new polymeric composites.

What are mass measurements good for?
To identify, verify, and quantitate: metabolites, recombinant
proteins, proteins isolated from natural sources, oligonucleotides,
drug candidates, peptides, synthetic organic chemicals, polymers

Assigning numerical value to the intrinsic property
of ―mass‖ is based on using carbon-12, 12C, as a
reference point.
One unit of mass is defined as a Dalton (Da).
One Dalton is defined as 1/12 the mass of a single
carbon-12 atom.
Thus, one 12C atom has a mass of 12.0000 Da.
How is mass defined?

Exact Masses of Some Common Elements and Their Isotopes:
Element Symbol Exact Mass (u) Rel. Abundance %
Hydrogen 1H 1.007825037 100.0
Deuterium 2H or D 2.014101787 0.015
Carbon 12 12C 12.00000 100.0
Carbon 13 13C 13.003354 1.11223
Nitrogen 14 14N 14.003074 100.0
Nitrogen 15 15N 15.00011 0.36734
Oxygen 16 16O 15.99491464 100.0
Oxygen 17 17O 16.9991306 0.03809
Oxygen 18 18O 17.99915939 0.20048
Fluorine 19F 18.998405 100.0
Sodium 23Na 22.9897697 100.0
Silicon 28 28Si 27.9769284 92.23
Silicon 29 29Si 28.9764964 5.0634
Silicon 30 30Si 29.9737717 3.3612
Phosphorus 31P 30.9737634 100.0
Sulfur 32 32S 31.972074 100.0
Sulfur 33 33S 32.9707 0.78931
Sulfur 34 34S 33.96938 4.43065
Sulfur 36 36S 35.96676 0.02105
Chlorine 35 35Cl 34.968854 100.0
Chlorine 37 37Cl 36.965896 31.97836

14
.
Mass Definitions
Molecular masses are measured in Daltons (Da) or mass units (u).
One Dalton = 1/12 of the mass of a 12C atom. The
International Union of Pure and Applied Chemistry (IUPAC) suggests the
unified atomic mass unit (abbreviated u), which is based on 12C . The
dalton (abbreviated Da) is identical in size to u
Monoisotopic mass = sum of the exact masses of the most abundant
isotope of each element present, i.e., 1H=1.007825, 12C=12.000000,
16O=15.994915, etc.
This is the most accurately defined molecular mass and is preferred if a
measurement of it can be determined.
Average mass = sum of the abundant averaged masses (―atomic weights‖)
of the constituent atoms of a given molecule.
The result is a weighted average over all of the naturally occurring isotopes
present in the compound. This is the common chemical molecular weight
that is used for stoichiometric calculations (H=1.0080, C=12.011, O=15.994,
etc.). The average mass cannot be determined as accurately as the
monoisotopic mass because of variations in natural isotopic abundances.

Vaccum system reduces collisions b/n
Ions and gas molecules.

Sample introduction-
Vaporize sample)
Ion source- ionize
Analyte gas
molecules
Mass analyzer:
Separates ions
According to M/Z
Count ions(Detect
Ions) A detector counts
the number of ions at
different deflections and
the data can be plotted as
a ‘spectrum’ of different
masses.

Mass Sor
Illustration of the basic components of a mass spectrometry system.
Solid
• Liquid
• Vapor
Ionization
Source
Mass
Analzyer
Detector
Inlet
Form ions
charged molecules
selected
ions
Data
System
Sort or separates
ions by M/Z
When ions strike
Detector it Detect ions
Block diagram that shows the basic parts of a mass spectrometer. The inlet
transfers the sample into the vacuum of the mass spectrometer. In the source region,
neutral sample molecules are ionized and then accelerated into the mass analyzer. The
mass analyzer is the heart of the mass spectrometer. This section separates ions,
either in space or in time, according to their mass to charge ratio. After the ions are
separated, they are detected and the signal is transferred to a data system for analysis.
High vacuum minimizes ion-molecule reactions, scattering, and neutralization of the ions.
Neutral sample
molecules

Different elements
can be uniquely
identified by their mass
MS principles

Basic principle:
MS basic principle is super, super, super simple ….
A charged particle moving in a magnetic field is deflected.
The ionized molecule is moving along some sort of magnetic field…
As the particle moves in to the magnetic field its path gets bent..
The degree of deflection depends up on the mass /charge(M/Z) or
mass to charge ratio.
In other words any given particle whether it has 20AMU or 1 charge or
40 AMU and 2 charges is going to
get deflect in to the same amount.
When a compound is exposed to a high voltage electric current it loses electrons and
gets converted to positively charged ions.
The basic magnetic or electric field acting on these moving charged ions (+ve ions)
Deflects them along circular path on a radius that is a function of their mass to charge
Ratio i.e. (m/z or m/e ratio)
M+
Ionized molecule
(Charged particle)
As the particle moves
In to the magnetic
Field its path gets bent
The degree of deflection depends
up on the M/Z ratio and get
Information about M/Z. Ultimately
We can figure out the mass.
Lighter components or components with more ionic charge will deflect in the field more than heavier or
less charged components

How basic technique works?
M
Ionized molecule is move
Along some sort of magnetic field
As the particles moves in the magnetic field its path gets bent.
The degree of deflection depends on mass to charge ratio( M/Z) .
Heavier particles deflects less (bend less)
more charge -----deflected more
Lighter particles deflects more.
The mass spectrum is a plot of relative intensities (which represent the ion abundances)on
the ordinate versus the m/z values of the ions on the abscissa.

The purpose of MS or spectroscopy is to provide clues about the structure of
Molecules.
In the chemistry test books you can see the picture of the molecules but you
Can’t see the picture in the test tube but you can get some clues
You have to see what is the types of clues we can get from mass spectrometry.
MS stats with the original molecule.
The original molecule is designaged as M (Capital M)
Capital M stands for original molecule.
In organic chemistry, a radical ion is typically indicated by a superscript
dot followed by the sign of the charge: and . In mass spectrometry,
the sign
is written first, followed by the superscripted dot.

M + 1e M.+ 2e+
MS starts with the original molecule (M) we call the original molecule capital M.
When we hit(whack) with high energy electron .
The high energy electron (does roughly speaking is it fetishize (with high
intensity) collides with a neutral analyte molecule can knocks off another
electron. (2e) resulting in a positively charged ion.
This high energy electron is going to knock another electron loose from a
molecule, so what should be the correct symbol of the molecule now
now that the molecule has electron not loose, is a radical and radicals with
unpaired electron and since it lost an electron it must have a +ve charge.
The symbol we would use is radical cation. A radical cation.
Original molecule
Radical cation or Molecular ion(M+)or parent ion.
M is the molecular ion produced by
removing a single electron to form a
radical cation. M is the molecule, + is
the charge of the cation, and (.) is the
remaining unpaired electron of the
radical.
High energy electrons smashing into sample molecules and
knocking electrons out of orbitals.

When the energy supplied is more than the ionized energy of the molecule, the
Molecular ion (M+) undergoes further fragmentation to give smaller ions and
Free radicals.
M+ A1
+ + A2 ( or ) A1 + A2
+

Ions are formed continuously in the ion source of mass spectrometer and
accelerated toward the detector by an electrical potential
Or the accelerating voltage V.
Once the accelerated ions then move in to the magnetic field B or H , whose
direction is perpendicular to their path.
The magnetic field is constrained to force the ions to follow a circular path (arc)
whose radius is r.
In a magnetic field, an ion with mass m will experience a centripetal
force is given by mv2/r(one pulling the ion toward the centre of the
circle) equal to Bzv
where B strength of the magnetic field, BZv = mv2/r
z is the charge on the ion,
Theory or concept of mass sectrometry (fragmentation)
mass spec equation for magnetic sector or basis for
Separation of particle (relation b/n m,r,V,and H equation)

From the equation it is clear that the radius of the circular path of a particle is
Depends on the magnetic field(H),accelerating voltage V and the m/z ratio.
The radius of the ionized molecule is dependent on mass (m) as the charge(e)
Voltage(V)and magnetic field (H) are constant.
The relation between mass(m),radius of the circular path(r) accelerating voltage (V)
And magnetic field(H) is shown in the above equation and is considered to be
The basis for separation of the particles with respect to their masses.

Chapter 12 30
Mass Spectrometer
=>

Atoms can be deflected by magnetic fields - provided the atom is first turned into an
ion. Electrically charged particles are affected by a magnetic field although
electrically neutral ones aren't.
The sequence is :
Stage 1: Ionisation
The atom is ionised by knocking one or more electrons off to give a positive ion. This
is true even for things which you would normally expect to form negative ions
(chlorine, for example) or never form ions at all (argon, for example). Mass
spectrometers always work with positive ions.
Stage 2: Acceleration
The ions are accelerated so that they all have the same kinetic energy.

Accelerating plates
Lets start out by looking at
This pair of electrodes.
Apply high voltage(potential)
Across them.its going to generate
High voltage electron beam.(High
Energy source of particles.
You can collide some sample that you
Are interested in.
Produce ions. Ions that we generate
Here will be accelerated towards either
-ve or +ve plates.
Ions starts moving in the different directions
+ve ions will be attrected towords -ve plates
-ve ions will be attracted towords + ve plates.
We are going to interested in the +ve ions only
+
-+
+-

Stage 3: Deflection
The ions are then deflected by a magnetic field according to their
masses. The lighter they are, the more they are deflected.
The amount of deflection also depends on the number of positive
charges on the ion - in other words, on how many electrons
were knocked off in the first stage. The more the
ion is charged, the more it gets deflected.
Stage 4: Detection
The beam of ions passing through the machine is detected
electrically.

Understanding what's going on
The need for a vacuum
It's important that the ions produced in the ionisation chamber have a free run
through the machine without hitting air molecules

The vaporised sample passes into the ionisation chamber. The electrically
heated metal coil gives off electrons which are attracted to the electron trap
which is a positively charged plate.
The particles in the sample (atoms or molecules) are therefore bombarded
with a stream of electrons, and some of the collisions are energetic enough to
knock one or more electrons out of the sample particles to make positive
ions.
Most of the positive ions formed will carry a charge of +1 because it is much
more difficult to remove further electrons from an already positive ion.
These positive ions are persuaded out into the rest of the machine by the ion
repeller which is another metal plate carrying a slight positive charge

The components of the mass spectrometer that cause ion formation,
separation,and detection are contained in an ultraclean housing usually kept
at moderately high vacuum (10-3–10-6 torr
High vacuum ensures that, once the ions formed in the ion source begin to
move toward the detector, they will not collide with other molecules
because this could result in further fragmentation or deflect them
from their desired path. Nearly all fragmentation reactions occurring
under these conditions are intramolecular (involving only the decomposition
of individual ions) rather than intermolecular (involving the reaction of ions
with other species that may be present).
High vacuum also protects the metal and oxide surfaces of the ion
source, analyzer, and detector from corrosion by air and water
vapor, which could compromise the spectrometer’s ability to form, separate,
and detect ions.
1 torr = 1 mm Hg, which is equivalent to 133 pascal
(Pa).

The positive ions are repelled away from
the very positive ionisation chamber and
pass through three slits, the final one of
which is at 0 volts. The middle slit carries
some intermediate voltage. All the ions are
accelerated into a finely focused beam
When an ion hits the metal box, its charge is neutralised by an electron
jumping from the metal on to the ion (right hand diagram). That leaves a space
amongst the electrons in the metal, and the electrons in the wire shuffle along to fill it.
A flow of electrons in the wire is detected as an electric current which can be
amplified and recorded. The more ions arriving, the greater the current

Different ions are deflected by the magnetic field by different amounts. The amount
of deflection depends on:
the mass of the ion. Lighter ions are deflected more than heavier ones.
the charge on the ion. Ions with 2 (or more) positive charges are deflected
more than ones with only 1 positive charge.
These two factors are combined into the mass/charge ratio.Mass/charge ratio is
given the symbol m/z (or sometimes m/e).
For example, if an ion had a mass of 28 and a charge of 1+, its mass/charge ratio
would be 28. An ion with a mass of 56 and a charge of 2+ would also have a
mass/charge ratio of 28.
Assuming 1+ ions, stream A
has the lightest ions, stream B
the next lightest and stream C
the heaviest. Lighter ions are
going to be more deflected
than heavy ones.

In the last diagram, ion stream A is most deflected - it will contain ions with the
smallest mass/charge ratio. Ion stream C is the least deflected - it contains ions
with the greatest mass/charge ratio.
It makes it simpler to talk about this if we assume that the charge on all the ions
is 1+. Most of the ions passing through the mass spectrometer will have a charge
of 1+, so that the mass/charge ratio will be the same as the mass of the ion.
Note: Youmustbeawareofthepossibilityof2+(etc)ions,butthevastmajorityofA'level questions
willgive youmassspectrawhichonlyinvolve1+ions.Unlessthereissomehintinthequestion, you
canreasonablyassumethattheionsyouaretalkingaboutwillhaveachargeof1+.
Assuming 1+ ions, stream A has the lightest ions,
stream B the next lightest and stream C the
heaviest. Lighter ions are going to be more
deflected than heavy ones.

DETECTION
Only ion stream B makes it right through the machine to the
ion detector. The other ions collide with the walls where
they will pick up electrons and be neutralised. Eventually,
they get removed from the mass spectrometer by the
vacuum pump.

Detecting the other ions
How might the other ions be detected - those in streams A and C which have been lost
in the machine?
Remember that stream A was most deflected - it has the smallest value of m/z (the
lightest ions if the charge is 1+). To bring them on to the detector, you would need to
deflect them less - by using a smaller magnetic field (a smaller sideways force).
To bring those with a larger m/z value (the heavier ions if the charge is +1) on to the
detector you would have to deflect them more by using a larger magnetic field.
If you vary the magnetic field, you can bring each ion stream in turn on to the detector to
produce a current which is proportional to the number of ions arriving. The mass of each
ion being detected is related to the size of the magnetic field used to bring it on to the
detector. The machine can be calibrated to record current (which is a measure of the
number of ions) against m/z directly. The mass is measured on the 12C scale.
Note: The 12C scale is a scale on which
the 12C isotope weighs exactly 12 units

What the mass spectrometer output looks like
The output from the chart recorder is usually simplified into a "stick diagram". This
shows the relative current produced by ions of varying mass/charge ratio.
The stick diagram for molybdenum looks lilke this:
You may find diagrams in which the vertical axis is labelled as either "relative
abundance" or "relative intensity". Whichever is used, it means the same thing. The
vertical scale is related to the current received by the chart recorder - and so to the
number of ions arriving at the detector: the greater the current, the more abundant
the ion.
As you will see from the diagram, the commonest ion has a mass/charge ratio of 98.
Other ions have mass/charge ratios of 92, 94, 95, 96, 97 and 100.
That means that molybdenum consists of 7 different isotopes. Assuming that the
ions all have a charge of 1+, that means that the masses of the 7 isotopes on the
carbon-12 scale are 92, 94, 95, 96, 97, 98 and 100
The largest peak in the mass
spectrum (100% relative
intensity) is called the base peak
This molecular ion (M+) is very important
because it has virtually the same mass as that of
the analyte molecule (the small mass of the lost
electron can be ignored).

The physics behind mass spectrometry is that a charged particle passing through a magnetic
field is deflected along a circular path on a radius that is proportional to the mass to charge
ratio, m/e.
In an electron impact mass spectrometer, a high energy beam of electrons is used to displace
an electron from the organic molecule to form aradical cation known as the molecular ion. If
the molecular ion is too unstable then it can fragment to give other smaller ions.
The collection of ions is then focused into a beam and accelerated into the magnetic field and
deflected along circular paths according to the masses of the ions. By adjusting the magnetic
field, the ions can be focused on the detector and recorded.

It is important to distinguish between the terms ions and peaks in
mass spectrometry. Ions are particles that have both mass
and charge, and they can fragment to form other ions. There can be
large or small numbers of ions, so that it is appropriate to speak of
their relative abundance.
On the other hand, peaks in a mass spectrum correspond to localized
maximum signals
produced by the detector and have only m/z values associated with
them. These signals are either weak or strong (depending on the
numbers of ions produced) and therefore are best described as having
intensity. The abundance of peaks implies that there are many peaks,
not that a given peak is big or little.

Mass Spectrometer Schematic
Inlet
Ion
Source
Mass
Filter
Detector
Data
System
High Vacuum System
Turbo pumps
Diffusion pumps
Rough pumps
Rotary pumps
Sample Plate
Target
HPLC
GC
Solids probe
MALDI
ESI
IonSpray
FAB
LSIMS
EI/CI
TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS
Microch plate
Electron Mult.
Hybrid Detec.
PC’s
UNIX
Mac

Inlet
Ion
source
Mass
Analyzer Detector
Data
System
High Vacuum System
Mass Spectrometer Block Diagram

Inlet
Ion
source
Mass
Analyzer Detector
Data
System
High Vacuum System
Mass Spectrometer Block Diagram
Turbo
molecular
pumps

Inlet
Ion
Source
Mass
Analyzer Detector
Data
System
High Vacuum System
HPLC
Flow injection
Sample plate
Sample Introduction

Inlet
Ion
Source
Mass
Analyzer Detector
Data
System
High Vacuum System
MALDI
ESI
FAB
LSIMS
EI
CI (APCI) atmospheric chemical
ionization.
Ion Source

Hard ion sources leave excess energy in molecule - extensive
fragmentation
Soft ion sources little excess energy in molecule - reduced
fragmentation

A) External (Batch) Inlet Systems:
Sample heated (<400 °C) in small external oven
Vapour admitted to ionizer through valve
Gas stream added to entrain analyte
(B) Direct Probe:
Sample vial inserted through air-lock into ionizer chamber
Vial heated to vaporize sample
Vial can be reduced to capillary or surface plate for small
quantities
(C) Chromatography/Electrophoresis/Injection Analysis
Can be modified to directly flow into ionizer region
Sample inlet system:

World War II. During this period,
Dempster developed EI.
EI was originally called electron
impact.
This is a process by which gas-phase
molecules at
a pressure of >10-3 Torr are ionized by
a beam of electrons, produced by a
hot wire (filament), that have
been accelerated by 70V (i.e. 70 eV).
EI is used in modern mass
spectrometers where analytes are in
the gas phase.
Electron Ionization (EI)

Also referred to as electron impact ionization, this is the oldest and best-characterized of
all the ionization methods. A beam of electrons passes through the gas-phase sample.
An electron that collides with a neutral analyte molecule can knock off another electron,
resulting in a positively charged ion.
The EI source is most commonly a small chamber about 1 cc in volume, in
which analyte molecules interact with a beam of highly energetic electrons that
have typically been accelerated through a potential difference of 50–70 volts (V)
across the volume of the ion source [50–70 electron volts (eV); 1 eV = 23 kcal].
This electron beam is produced by boiling electrons off a narrow strip or coil of
wire made of a tungsten-rhenium alloy. Between the filament and the center of
the ion source is a metal plate with a slit called the electron aperture. This slit limits
the size of the electron beam and confines ionization to a small volume within the
center of the ion source. Opposite the filament is the collector, a metal plate held at
a positive electrical potential (+V) that attracts and intercepts the electron
beam after it has passed through the source. a collimating magnet, which causes
the electrons in the beam to travel in a helical path. Although this helical trajectory
improves the probability that the electrons and molecules will interact, sample
ionization
is still very inefficient—less than one molecule in a thousand undergoes
ionization.
What happens during ionization is complex. electrons
smashing into sample molecules and knocking electrons out of orbitals.

one electron is ejected from one of the bonding or nonbonding orbitals of the molecule
Ionization energies (IE) for most organic compounds range from about 5–15 eV.
Bond dissociation energies are even smaller, so this method of ionization not only
causes molecules to expel one or more electrons, it also provides enough energy for
substantial fragmentation of the first-formed ion (the molecular ion, M+).
Because of the excess energy present in 50–70 eV electrons, enough additional
energy may be transferred to overcome the second, or even third, ionization
potential of the molecule, leading to ions having +2 or +3 charges.
Many different products form during ionization.
Excited molecules can return to their neutral ground state
through thermal vibrations or the emission of light, and because
no ions are formed in the process, they are simply pumped away
from the ion source by the vacuum System.

Sometimes the analyte molecule absorbs an electron and a negative ion is
formed (Table 1.2, product b). In order to be absorbed by the molecule, the electron
must be of very low energy (0.1 eV), and there are few electrons of this energy in
a standard EI source. By reversing the polarity of the repeller, ion focusing plate,
and extractor plate in the ion source, and by altering the detector so that it will
detect negative ions, a negative ion mass spectrum can be recorded. For most
compounds
negative ion MS offers few advantages over positive ion MS, and overall it
tends to be less sensitive. There are some specific applications, however, most
notably with halogenated compounds. In this book only positive ion products and
their fragmentations will be covered.
The remaining products listed in Table 1.2 are positive ions. The ion that is
formed first results directly from ejection of a single electron from the neutral molecule
(product c). This molecular ion (M+) is very important because it has virtually
the same mass as that of the analyte molecule (the small mass of the lost
electron can be ignored). Indeed, mass spectrometry is one of the few analytical
tools available for determining the molecular mass of a compound.

Ion products d and e in Table 1.2 are formed by unimolecular dissociation of
M+. In the first case a single bond is broken and a neutral group of atoms
having an odd number of electrons (called a radical; is lost.
The second process (dissociation with rearrangement) involves breaking
some bonds while attach same time forming new ones. This results in
expulsion of a fragment containing an even number of electrons, usually as a
neutral molecule.
Table 1.2 imply that such ions are formed in a concerted process in which
ionization, bond making, and bond breaking all occur at about the same time.
However, fragmentations that involve rearrangement of atoms usually occur in
a stepwise fashion through one or more intermediates.
If more than one electron is ejected from the analyte molecule, ions having
charges of +2, +3, or even +4 may be formed (Table 1.2, products f ).
Biopolymers such as peptides may have charge states of +10 or more from
protonation of basic sites on the molecule. Since mass spectrometry actually
measures the mass-to charge ratio (m/z) of an ion, not its mass, an ion having
a charge greater than +1 is found not at the m/z value corresponding to its
mass (m), but rather at m/2, m/3, or m/4, depending on the number of charge
states. Further, if m is not evenly divisible by the number of charges z, m/z will
have a non integral value. For example, the double charged molecular ion
(M2+) of a compound having a molecular mass of 179 is found at m/z 179/2 =
89.5.

Neutral products are removed by the vacuum system, because the electric and
magnetic fields present in the ion source have no effect on their motion.
Positive and negative ions, on the other hand, can be separated by appropriately
placed charged surfaces
in the ion source (Figure 1.2). To accomplish this, the repeller is kept at a positive
potential (+V) both to attract and neutralize negative ion products and to repel positive
ions. Conversely, the extractor plate and ion focusing plate (the ion optics) are
both kept at a negative electrical potential (V) to attract and accelerate the positive
ions toward the m/z analyzer. Slits in the extractor and ion focusing plates allow
passage of the positive ions and help focus the ion beam as it approaches the analyzer

In the first case a single bond is
broken and a neutral group of
atoms having an odd number of
electrons (called a radical; is lost
breaking some bonds while
attach same time forming new
ones. This results in expulsion
of a fragment containing an
even number of electrons,
usually as a neutral moleculeIf more than one electron is ejected from
the analyte molecule, ions having charges
of +2, +3, or even +4 may be formed

Chemical Ionization
Unlike EIMS, in which molecules are ionized through interaction with high-energy
electrons, ionization in chemical ionization mass spectrometry (CIMS) depends on
collisions of ions and molecules. In positive ion CIMS the sample is ionized by
reaction with ions generated within a large excess of a relatively low molecular
mass reagent gas such as methane (as CH+5 ), isobutane [as (CH3)3C+], or ammonia
(as NH+4), at a pressure of about 1 torr. Although some reagent gas ions are
themselves formed by ion/molecule reactions

This type of ion formation (often called soft ionization) imparts significantly less
energy to analyte molecules than do interactions with high-energy electrons, so that
the resulting ions have little excess internal energy. These ions therefore fragment
less than those formed by EIMS. As a result, although CIMS is useful for determining
the molecular mass of compounds that do not produce a detectable M+. by
EIMS

Fast Atom Bombardment and Secondary Ion Mass Spectrometry. (5) Fast Atom
Bombardment (FAB) and Secondary Ion Mass Spectrometry (SIMS) both use high
energy atoms
to sputter and ionize the sample in a single step. In these techniques, a beam of rare
gas neutrals
(FAB) or ions (SIMS) is focused on the liquid or solid sample. The impact of this high
energy
beam causes the analyte molecules to sputter into the gas phase and ionize in a single
step
(Figure 6). The exact mechanism of this process is not well understood, but these
techniques
work well for compounds with molecular weights up to a few thousand dalton. Since no
heating
is required, sputtering techniques (especially FAB) are useful for studying thermally
labile
compounds that decompose in conventional inlets (6, 7).

Fast atom bombardment:
The most significant difference between FAB and SIMS is the sample preparation. In
FAB the analyte is dissolved in a liquid matrix. A drop of the sample/matrix mixture is
placed at the end of an insertion probe and introduced to the source region. The fast
atom beam is focused on this droplet to produce analyte ions. Glycerol or similar low
vapor pressure liquids are typically used for the matrix. Ideally, the analyte is soluble
in the liquid matrix and a monolayer of analyte forms on the surface of the droplet

SIMS experiments(8) are used to study surface species and solid samples.* No
matrix is
used and the ionizing beam is focused directly on the sample. Although this makes
sampling
more difficult, it is useful for studying surface chemistry. High resolution chemical
maps are
produced by scanning a tightly focused ionizing beam across the surface and depth
profiles are
produced by probing a single location(9, 10). Although SIMS is a very sensitive and
powerful
technique for surface chemistry and materials analysis, the results are often difficult
to quantitate

Sample introduction
. heated batch inlet
. heated direct insertion probe
. gas chromatograph
. liquid chromatograph (particle-beam interface)
Benefits
. often gives molecular weight information through molecular-like ions such as
[M+H]+,
even when EI would not produce a molecular ion.
. simple mass spectra, fragmentation reduced compared to EI
Limitations
sample must be thermally volatile and stable
. less fragmentation than EI, fragment pattern not informative or reproducible enough
for library search
. results depend on reagent gas type, reagent gas pressure or reaction time, and
nature of sample.
Mass range
. Low Typically less than 1,000 Da.

Desorption Chemical Ionization (DCI)
Summary
This is a variation on chemical ionization in which the analyte is placed on a filament
that is rapidly heated in the CI plasma. The direct exposure to the CI reagent ions,
combined with the rapid heating acts to reduce fragmentation. Some samples that
cannot be thermally desorbed without decomposition can be characterized by the
fragments produced by pyrolysis DCI.
Sample introduction
. sample deposited onto a filament wire
. filament rapidly heated inside the CI source.
Benefits
. reduced thermal decomposition
. rapid analysis
. relatively simple equipment
Limitations
. not particularly reproducible
. rapid heating requires fast scan speeds
fails for large or labile compounds
Mass range
Low Typically less than 1,500 Da.

Negative-ion chemical ionization (NCI)
Summary
Not all compounds will produce negative ions. However, many important compounds of
environmental or biological interest can produce negative ions under the right
conditions. For such
compounds, negative ion mass spectrometryis more efficient, sensitive and selective
than positive-ion mass spectrometry.
Negative ions can be produced by a number of processes. Resonance electron
capture refers to the capture of an electron by a neutral molecule to produce a
molecular anion. The electron energy
is very low, and the specific energy required for electron capture depends on the
molecular structure of the analyte.
Electron attachment is an endothermic process, so the resulting molecular anion will
have excess energy. Some molecular anions can accommodate the excess energy.
Others may lose the electron or fall apart to produce fragment anions. Electron
attachment is an endothermic process, so the resulting molecular anion will have
excess
energy. Some molecular anions can accommodate the excess energy. Others may
lose the electron
or fall apart to produce fragment anions.
In negative-ion chemical ionization, a buffer gas (usually a common CI gas such as
methane) is
used to slow down the electrons in the electron beam until some of the electrons have
just the

BENEFITS
efficient ionization, high sensitivity
. less fragmentation than positive-ion EI or CI
. greater selectivity for certain environmentally or biologically important compounds
Limitations
. not all volatile compounds produce negative ions
. poor reproducibility
Mass range
. Low Typically less than 1,000 Da.
Field

Field Desorption and Ionization
These methods are based on electron tunneling from an emitter that is biased at a high
electrical
potential. The emitter is a filament on which fine crystalline 'whiskers' are grown. When a
high
potential is applied to the emitter, a very high electric field exists near the tips of the
whiskers.
There are two kinds of emitters used on JEOL mass spectrometers: carbon emitters and
silicon
emitters. Silicon emitters are robust, relatively inexpensive, and they can handle a higher
current
for field desorption. Carbon emitters are more expensive, but they can provide about an
order of
magnitude better sensitivity than silicon emitters.
Field desorption and ionization are soft ionization methods that tend to produce mass
spectra
with little or no fragment-ion content.

Field Desorption (FD)
Summary
The sample is deposited onto the emitter and the emitter is biased to a high potential
(several
kilovolts) and a current is passed through the emitter to heat up the filament. Mass
spectra are
acquired as the emitter current is gradually increased and the sample is evaporated
from the emitter
into the gas phase. The analyte molecules are ionized by electron tunneling at the tip
of the
emitter 'whiskers'. Characteristic positive ions produced are radical molecular ions
and cationattached
species such as [M+Na]+ and [M-Na]+. The latter are probably produced during
desorption
by the attachment of trace alkali metal ions present in the analyte

Sample introduction
Direct insertion probe.
The sample is deposited onto the tip of the emitter by
. dipping the emitter into an analyte solution
. depositing the dissolved or suspended sample onto the emitter with a microsyringe
Benefits
. simple mass spectra, typically one molecular or molecular-like ionic species per
compound.
. little or no chemical background
. works well for small organic molecules, many organometallics, low molecular weight
polymers and some petrochemical fractions
Limitations
. sensitive to alkali metal contamination and sample overloading
. emitter is relatively fragile
. relatively slow analysis as the emitter current is increased
. the sample must be thermally volatile to some extent to be desorbed
Mass range
. Low-moderate, depends on the sample. Typically less than about 2,000 to 3,000 Da.
. some examples have been recorded from ions with masses beyond 10,000 Da.

Field Ionization (FI)
Summary
The sample is evaporated from a direct insertion probe, gas chromatograph, or gas
inlet. As the
gas molecules pass near the emitter, they are ionized by electron tunneling.
Sample introduction
. heated direct insertion probe
. gas inlet
. gas chromatograph
Benefits
. simple mass spectra, typically one molecular or molecular-like ionic species per
compound.
. little or no chemical background
. works well for small organic molecules and some petrochemical fractions
Limitations
The sample must be thermally volatile. Samples are introduced in the same way as for
electron ionization (EI).
Mass range
. Low Typically less than 1000 Da.

Different Ionization Methods
• Electron Impact (EI - Hard method)
– small molecules, 1-1000 Daltons, structure
• Fast Atom Bombardment (FAB – Semi-hard)
– peptides, sugars, up to 6000 Daltons
• Electrospray Ionization (ESI - Soft)
– peptides, proteins, up to 200,000 Daltons
• Matrix Assisted Laser Desorption (MALDI-Soft)
– peptides, proteins, DNA, up to 500 kD

86
++
+
+
+
+
+
+
+ +
+
+
+
+
+ +
+
+
+
++
+
++
+
++
+
+
+
+
+
+
+
+
+
+
++
+
++
++
+
MH+
[M+2H]2+
[M+3H]3+
Electrospray Ion Formation
Droplets formed in electric field have excess positive ions.
Evaporation of neutrals concentrates charge.
Droplets break into smaller droplets.
Eventually one molecule + n protons is left.
+
+
+
+++
+
++
++++
Needle at High Voltage
+
+
+
++
++
+ +
+
+
+ +++
+

High voltage applied
to metal sheath (~4 kV)
Sample Inlet Nozzle
(Lower Voltage)
Charged droplets
++
+
+
+
+
+
+
++
+
+
+
+
++
+
+ +
++
+
++
+
++
+++
+
+
+
+
+
+
+
+
++
+
++
++
+
MH+
MH3
+
MH2
+
Pressure = 1 atm
Inner tube diam. = 100 um
Sample in solution
N2
N2 gas
Partial
vacuum
Electrospray ionization:
Ion Sources make ions from sample molecules
(Ions are easier to detect than neutral molecules.)

Most of the molecules don’t have a charge on them.
Generally magnetic force effects the charge particles. A mass spectrometer works
by using magnetic and electric fields to exert forces on charged particles
(ions) in a vacuum. Therefore, a compound must be charged or ionized
to be analyzed by a mass spectrometer.
The first question is
How do you get it charged on the molecules
Historically the first technique developed was called EI(Electron impact ionization)
EIMS
The basic idea is
The electron gun is used to ionized the molecule
You give it a good whack, you knock an electron you get a Molecular cation.
For ex
CH4 + e- CH4+. +2e-
Methane and you hit with an electron. You just take an electron out of it
You get a radical cation .What mass spectrometry called a molecular ion.

just prior toWorld War II. During this period, Dempster
developed EI. [EI was originally called electron impact.
This is a process by which gas-phase molecules at
a pressure of >10-3Torr are ionized by a beam of
electrons, produced by a hot wire (filament), that have
been accelerated by 70V (i.e. 70 eV).] EI is used in
modern mass spectrometers where analytes are in the
gas phase.

Several common modes differing by method of ion formation:
Electrospray (ESI)
Atmospheric Pressure Chemical Ionization (APCI)
Atmospheric Pressure Photo-Ionization (APPI)
New dual sources (ESI/APCI) or (APCI/APPI)
Which is best?
It depends on the exact application.
Increasing polarity and molecular weight and
thermal instability favors electrospray.
Most drugs of abuse are highly polar
and are easily analyzed using
electrospray.
High molecular weight proteins also
require electrospray
Lower polarity and molecular weight favors
APCI or APPI.
Lower background, but compounds
must be more thermally stable.

Electrospray is a method of getting the solution phase ions into the gas phase so that they
can be sampled by the mass spectromete
Three Fundamental Processes:
1. Production of charged droplets.
2. Droplet size reduction, and fission.
3. Gas phase ion formation

Orifice
1. A large voltage ( up to 6kV) is applied between the end of a capillary carrying the
LC mobile phase and the mass spectrometer entrance.
2. Ions (of the same polarity) are drawn out toward the counter electrode (curtain
plate) pulling the mobile phase along.
3. When the excess charge at the tip of the capillary overcomes surface tension, a
droplet is formed.

Electrospray ionization
Eluent is sprayed (nebulized) into a
chamber at atmospheric pressure in the
presence of a strong electrostatic field
and heated drying gas.
The electrostatic field causes further
dissociation of the analyte molecules.
The heated drying gas causes the solvent
in the droplets to evaporate. As the droplets
shrink, the charge concentration in the
droplets increases.
Eventually, the repulsive
force between ions with like charges exceeds
the cohesive forces and ions are ejected
(desorbed) into the gas phase. These ions
are attracted to and pass through a capillary
sampling orifice into the mass analyzer.

Electrospray is especially useful for analyzing
large biomolecules such as proteins, peptides,
and oligonucleotides,
but can also analyze
smaller molecules
like benzodiazepines
and sulfated
conjugates.
electrospray can
be used to analyze
molecules as large
as 150,000 u
a typical LC/MS
instruments is around 3000 m/z. For example:
100,000 u / 10 z = 1,000 m/z
When a large molecule acquires many charges,
a mathematical process called deconvolution
is often used to determine the actual molecular weight of an analyte.

In atmospheric pressure ionization, the analyte molecules are ionized first, at atmospheric
pressure. The analyte ions are then mechanically and electrostatically separated from neutral
molecules. Common atmospheric pressure ionization techniques are
• Atmospheric pressure chemical ionization (APCI)
• Atmospheric pressure photoionization (APPI)
Atmospheric pressure chemical ionization
In APCI, the LC eluent is sprayed through a heated (typically 250°C – 400°C) vaporizer
at atmospheric pressure. The heat vaporizes the liquid. The resulting gas-phase solvent
molecules are ionized by electrons discharged from a corona needle. The solvent ions
then transfer charge to the analyte molecules through chemical reactions (chemical
ionization).
The analyte ions pass through a capillary sampling orifice into the mass analyzer.
APCI is applicable to a wide range of polar and nonpolar molecules. It rarely results in
multiple charging so it is typically used for molecules less than 2000 u. Due to this,
and because it involves high temperatures, APCI is less well-suited than electrospray
for analysis of large biomolecules that may be thermally unstableMass range
. Low-moderate Typically less than 2000 Da.
Benefits
. good for less-polar compounds
. excellent LC/MS interface
. compatible with MS/MS methods

Atmospheric pressure photoionization
Atmospheric pressure photoionization (APPI)
for LC/MS is a relatively new technique. As
in APCI, a vaporizer converts the LC eluent to
the gas phase. A discharge lamp generates
photons in a narrow range of ionization
energies. The range of energies is carefully
chosen to ionize as many analyte molecules
as possible while minimizing the ionization
of solvent molecules. The resulting ions pass
through a capillary sampling orifice into
the mass analyzer.
APPI is applicable to many of the same
compounds that are typically analyzed by
APCI. It shows particular promise in two
applications, highly nonpolar compounds
and low flow rates (<100 ìl/min), where APCI
sensitivity is sometimes reduced.
In all cases, the nature of the analyte(s)
and the separation conditions have a strong
influence on which ionization technique:
electrospray, APCI, or APPI, will generate
the best results. The most effective technique
is not always easy to predict.

Terminology
Molecular ion The ion obtained by the loss of an electron from the molecule
Base peak The most intense peak in the MS, assigned 100% intensity
M+
Symbol often given to the molecular ion
Radical cation +
ve charged species with an odd number of electrons
Fragment ions
Lighter cations formed by the decomposition of the molecular ion.
These often correspond to stable carbcations.
Spectra
The MS of a typical hydrocarbon, n-decane is shown below. The molecular ion is
seen as a small peak at m/z = 142. Notice the series ions detected that correspond
to fragments that differ by 14 mass units, formed by the cleaving of bonds at
successive -CH2- units

Matrix-Assisted Laser Desorption Ionization (MALDI)
Summary
The analyte is dissolved in a solution containing an excess of a matrix such as sinapinic acid
or
dihydroxybenzoic acid that has a chromophore that absorbs at the laser wavelength. A
small amount of this solution is placed on the laser target. The matrix absorbs the energy
from the laser pulse and produces a plasma that results in vaporization and ionization of
the analyte.
Sample introduction
. direct insertion probe
. continuous-flow introduction
Benefits
. rapid and convenient molecular weight determination
Limitations
. MS/MS difficult
. requires a mass analyzer that is compatible with pulsed ionization techniques
. not easily compatible with LC/MS
Mass range
. Very high Typically less than 500,000 Da.

Direct laser desorption relies on the very rapid heating of the sample or sample
substrate to vaporize molecules so quickly that they do not have time to
decompose. This is good for low to medium-molecular weight compounds and
surface analysis. The more recent development of matrix-assisted laser
desorption ionization (MALDI) relies on the absorption of laser energy by a
matrix compound. MALDI has become extremely popular as a method for the
rapid determination of high-molecular-weight compounds.
The analyte is dissolved in a solution containing an excess of a matrix such as
sinapinic acid or dihydroxybenzoic acid that has a chromophore that absorbs at
the laser wavelength. A smallamount of this solution is placed on the laser target. The
matrix absorbs the energy from the laser
pulse and produces a plasma that results in vaporization and ionization of the analyte.
Sample introduction
. direct insertion probe
. continuous-flow introduction
Benefits
. rapid and convenient molecular weight determination
Limitations
. MS/MS difficult
. requires a mass analyzer that is compatible with pulsed ionization techniques
. not easily compatible with LC/MS
Mass range
. Very high Typically less than 500,000 Da.

Sinapinic acid -cyano-4-hydroxycinnamic acid (CHCA) 2,5-dihydroxybenzoic acid (DHB)
HO
COOH
OHCH3O
CH3O
HO CH=CH-COOH HO CH=C-COOH
CN
Analyte is dissolved in solution with excess
matrix (>104).
Sample/matrix mixture is dried on a target and
placed in the MS vacuum.
Requirements for a satisfactory matrix:
It must co-crystallize with typical analyte
molecules
It must absorb radiation at the wavelength of
the laser (usually 337 nm)
To transfer protons to the analyte it should be
acidic
Typical successful matrices for UV MALDI are
aromatic carboxylic acids.

MALDI: Matrix Assisted Laser Desorption/Ionization.
The sample is prepared by mixing the analyte
and a matrix(Sinapinicacid,Dihydroxybenzoic
acid that has a chromophore that absorbs at the
laser wave length) compound chosen to absorb
the laser wavelength. This is placed on a probe
tip and dried.
A laser beam is then focused on this dried
mixture and the energy from a laser pulse is
absorbed by the matrix
The matrix absorbs the energy from the laser pulse
and produces a plasma that results in vaporization
and ionization of the analyte.
.(MALDI) is used to analyze extremely large molecules .
MALDI is often used for the analysis of synthetic and natural polymers, proteins, and
peptides. Analysis of compounds with molecular weights up to 500,000 dalton is possible.
Desorbed sample
ions and neutrals
Pulsed laser (337 nm)

105
±20 kV
Sample and matrix,
crystallized on stage
Desorbed sample
ions and neutrals
Pulsed laser
(337 nm)
3.5 ns
Sample stage
Mass analyzer
Matrix-assisted laser desorption ionization (MALDI)

106
-
+
-
+
+
++
--
-
--
-
-
--
-
-
-
-
-
+
+
+
+
+
+
++
+
+
+
1. Laser pulse produces matrix neutrals, + and - ions, and
sample neutrals: M --> M*, MH+, (M-H)- (M= Matrix)
2. Sample molecules are ionized by gas-phase proton transfer:
MH+ + A --> AH+ + M (A=Analyte)
(M-H)- + A --> (A-H)- + M
MALDI Ionization Mechanism

107
Mass (m/z) analyzers can be divided into two broad
categories: (1) those that in some way isolate ions
of individual m/z values from a beam – beam-type
instruments; and (2) those that store ions of all m/z
values and detect ions through some process of single m/z
isolation – traps. Magnetic-sector, double-focusing, TQ,
and TOF mass spectrometers are beam-type instruments.
QIT (both external and internal ionization variations)
and ICR mass spectrometers are traps.
the mass spectrometer used to separate
gas-phase ions according to their m/z
values is the mass analyzer. Mass
analyzer is the traditional terminology.

108
(A) Magnetic Sector Analyzers:
Magnetic-sector mass spectrometers use
only a magnetic
field to separate ions according to their m/z
values
. These instruments are referred to as
single-focusing mass spectrometers. They
are capable of
separating ions that differ in one m/z unit
over a range
from 1 to 700m/z.

Quadrupole Mass Analyzer
Uses a combination of RF
and DC voltages to operate
as a mass filter.
• Has four parallel metal
rods.
• Lets one mass pass
through at a time.
• Can scan through all
masses or sit at one
fixed mass.

110
A quadrupole mass analyzer consists of four parallel rods arranged in a square.
The analyte ions are directed down the center of the square.
Voltages applied to the rods generate electromagnetic fields.
These fields determine which mass-to-charge ratio of ions can pass
through the filter at a given time.
Quadrupoles tend to be the simplest and least expensive mass analyzers.
Quadrupole mass analyzers can operate in
two modes:
• Scanning (scan) mode
• Selected ion monitoring (SIM) mode
The analyzer consists of four rods or electrodes arranged across
from each other . As the ions travel through the quadrupole they are filtered
according to their m/z value so that only a single m/z value ion can strike the
detector. The m/z value transmitted by the quadrupole is determined by the
Radio Frequency (RF) and Direct Current (DC) voltages applied to the electrodes.
These voltages produce an oscillating electric field that functions as a bandpass
filter to transmit the selected m/z value.
Quadrupole mass analyzer

111
In scan mode, the mass analyzer monitors a range of mass-
to-charge ratios.
In SIM mode, the mass analyzer monitors only a few mass
to-charge ratios.
SIM mode is significantly more sensitive than scan mode
but provides information about fewer ions. Scan mode is
typically used for qualitative analyses or for quantitation
When all analyte masses are not known in advance.
SIM mode is used for quantitation and monitoring of target
compounds.

mass scanning mode
m1m3m4 m2
m3
m1
m4
m2
single mass transmission mode
m2 m2 m2 m2
m3
m1
m4
m2
Quadrupoles have variable ion transmission modes

CID and multiple-stage MS
Multiple-stage MS (also called tandem MS or MS/MS or MSn) is a powerful
way to obtain structural information. In triple-quadrupole or
quadrupole/quadrupole/time-of-flight instruments
the first quadrupole is used to select the precursor ion.
CID(Collision-Induced Dissociation) takes place in the second stage (quadrupole or
octopole), which is called the collision cell.
The third stage (quadrupole or TOF) then generates a spectrum of the resulting
product ions.
It can also perform selected ion monitoring of only a few product ions
when quantitating target compounds
To obtain structural information, analyte ions are fragmented by colliding them
with neutral molecules in a process known as collisioninduced
dissociation (CID) or collisionally activated dissociation (CAD)

114
The mass analyzer is the heart of the mass spectrometer. This section
separates ions, either in space or in time, according to their mass to
charge ratio.

116
Double-focusing Mass Spectrometer
Double-focusing mass spectrometers use a magnetic field
to select ions based on theirm/z values and an electric field
to select ions based on their energy. These instruments
became the workhorse of MS from the 1930s through
the end of the 1970s. These instruments are capable of
separating ions with very small differences in m/z values
allowing for the determination of the elemental composition
of the ion based on these millimass measurements.
CEC was the first commercial manufacturer of doublefocusing
mass spectrometers beginning before World war II

118
Quadrupole mass analyzers are often called mass filters because of the similarity
between m/z selection by a quadrupole and wavelength selection by an optical filter or
frequency selection by an electronic filter.
MASS ANALYZERS:
After ions are formed in the source region they are accelerated into the mass analyzer
by an electric field. The mass analyzer separates these ions according to their m/z
value. The selection of a mass analyzer depends upon the resolution,** (26) mass
range,*** scan rate**** and detection limits
Analyzers are typically described as either continuous or pulsed. Continuous analyzers
include quadrupole filters and magnetic sectors.
Pulsed analyzers include time-of-flight, ion cyclotron resonance, and quadrupole ion
trap mass spectrometers.
They transmit a single selected m/z to the detector
and the mass spectrum is obtained by scanning the analyzer so that different mass to
charge ratio ions are detected.
These (pulsed)instruments collect an entire mass spectrum from a single pulse of ions.
This results in a signal to noise advantage similar to Fourier transform or multichannel
spectroscopic techniques

119
Time-of-flight (TOF)
In a time-of-flight (TOF) mass analyzer,
a uniform electromagnetic force
is applied to all ions at the same time,
causing them to accelerate
down a flight tube.
Lighter ions travel faster and
arrive at the detector first,
so the mass-to-charge ratios
of the ions are determined by
their arrival times.
Time-of flight mass analyzers have
a wide mass range and can be very
accurate in their mass measurements.

122
time-of-flight mass
spectrometer (TOF-MS). Ions of different m/z values
accelerated from a region such as an ion source into
an evacuated tube will have different velocities, and
therefore these ions will reach the end of this evacuated
region at different times. By separating the times at which
ion current is observed at a detector placed at the end
of this evacuated region, it is possible to obtain a mass
spectrum. Ions of the lowest m/z will reach the detector
First.

124
Applications of mass
spectrometry include identifying and quantitating pesticides in water samples, it
identifying
steroids in athletes, determining metals at ppq (Parts Per Quadrillion) levels in water
samples,
carbon-14 dating the Shroud of Turin using only 40 mg of sample (1), looking for life on
Mars,
determining the mass of an 28Si atom with an accuracy of 70 ppt(2), and studying the
effect of
molecular collision angle on reaction mechanisms.
Mass spectrometry is essentially a technique for "weighing" molecules.* Obviously, this
is not done with a conventional balance or scale. Instead, mass spectrometry is based
upon the
motion of a charged particle, called an ion, in an electric or magnetic field. The mass to
charge
ratio (m/z)** of the ion effects this motion. Since the charge of an electron is known, the
mass to
charge ratio a measurement of an ion's mass.

Time-of-flight (TOF) Mass Analyzer
+
+
+
+
Source Drift region (flight tube)
detector
V
• Ions are formed in pulses.
• The drift region is field free.
• Measures the time for ions to reach the detector.
• Small ions reach the detector before large ones.

126
+
+
+
+
Source Drift region (flight tube)
detector
V
•Ions formed in pulses.
•Measures time for ions to reach the detector.
Time-of-Flight (TOF) Mass Analyzer
2
2
2
L
Vt
zm or zmt

127
Quadrupole Ion Trap
•Uses a combination of DC
and RF fields to trap ions
•Ions are sequentially
ejected by scanning the
RF voltage
Linear Trap
•Essentially a quadrupole with end-caps
•Advantage: Larger ion storage capacity, leading to better dynamic range
Ions in
(from ESI)
3D Trap
End caps
Ions out
to detector
Ring electrode
(~V)
Insulated
spacer
He gas
1x10-3 Torr
Raymond E. March, JOURNAL OF MASS SPECTROMETRY, VOL. 32, 351È369 (1997)

128
Ion trap
An ion trap mass analyzer consists of
a circular ring electrode plus two end
caps that together form a chamber. Ions
entering the chamber are ―trapped‖ there
by electromagnetic fields. Another field
can be applied to selectively eject ions
from the trap.
Ion traps have the advantage of being able
to perform multiple stages of mass
spectrometry
without additional mass analyzers

129
Electron Multiplier
From Detector Technolgy: http://www.detechinc.com/ B. Brehm et al., Meas. Sci. Technol. 6 (1995) 953-958.
Multi-Channel Plate (MCP)

130
Fourier transform-ion
cyclotron resonance (FT-ICR)
An FT-ICR mass analyzer (also called FT-MS)
is another type of trapping analyzer.
Ions entering a chamber are trapped in circular
orbits by powerful electrical and magnetic
fields.
When excited by a radio-frequency
(RF) electrical field, the ions generate a time
dependent current.
This current is converted by Fourier transform
into orbital frequencies of the ions which
correspond to their mass-to charge ratios.
Like ion traps, FT-ICR mass analyzers can
perform multiple stages of mass spectrometry
without additional mass analyzers.
They also have a wide mass range and excellent
mass resolution.
They are, however, the most expensive of
the mass analyzers.

131
B0
Detect
++
+
+
+
+
+
+
+
R C
Excite
+
+
+
+
+
+
+
+
+
Fourier Transform Ion Cyclotron Resonance (FT-ICR)
•Ions trapped and
measured in ultrahigh vacuum
inside a superconducting magnet.
A.G. Marshall
zm
1

132
Differential
Amplifier
FT
100 150 200 250
Frequency (kHz)
7+
8+
10+
11+
12+
9+
600 1000 1400 1800
12+
11+
10+
9+
8+
7+
m/z
Calibration
0
80 240 400
Time (ms)
Image
Current
Bovine
Ubiquitin
10721071
Fourier Transform Ion Detection
A.G. Marshall

134
Comparison of Analyzer Types
Ion Trap/
Quadrupole
TOF OrbiTrap FT-ICR
Sensitivity +++ ++* to +++ ++* +*
Mass
Accuracy
+** ++ +++ +++**
Resolving
Power
+** ++ +++ ++++**
Dynamic
Range
+ to +++** ++ +++ ++**
Upper m/z + ++++ +++ ++
*Sensitivity lowered due to losing ions on way to analyzer, rather than inherent
sensitivity.
**Can be improved by scanning narrower mass range or slower.

135
Alexander Makarov, Anal. Chem. 2000, 72, 1156-1162
Orbitrap
TOF
•Simultaneous excitation
FTICR
•Confined ion trajectory
•Image current detection
•Fourier transform data conversion
Unique to Orbitrap
•3D electric field trapping
•No need for magnet
•Easy access
•Final detection device

136
Image Current Detection in Orbitrap
From Alexander Makarov‘s 2008 ASMS Award Address

137
Three Important Properties to Assess Performance of
a Mass Spectrometer
1. Sensitivity
•Minimum quantity of sample needed (always estimate how much
sample you have, in femtomoles!)
2. Mass Accuracy
•Needed for identifying samples
by database searching or to determine elemental composition
3. Resolving Power
•Determine charge state. Resolve mixtures. High resolving can
also improve mass accuracy.

138
Mass (Measurement) Accuracy
Mass Accuracy or Mass Measurement Error is the difference between the
experimental mass (Mexp) and the theoretical value (Mtheo), calculated from
elemental composition.
In absolute term, , in Da or milli-Da
In relative term, , unit-less (ppm for high resolution MS)
Example:
Mexp = 1569.684
Mtheo= 1569.66956
Mass Measurement Error = 0.014Da or 9.2ppm
theo
theo
M
MM
MA exp
theo
MMMA exp
http://physics.nist.gov/PhysRefData/Elements/per_noframes.html

15.01500 15.01820 15.02140 15.02460 15.02780 15.03100
Mass (m/z)
100
0
10
20
30
40
50
60
70
80
90
100
%Intensity
ISO:CH3
15.0229
M
FWHM = M
R = M/ M
How is mass resolution calculated?

140
Resolving Power
•Measure of the ability to differentiate between components of similar
mass.
•Two definitions:
•Valley Definition: Neighboring peaks overlap at 10% peak apex height.
•Full Width Half Maximum (FWHM): Width of a single peak measured at
50% peak apex. This is the most commonly used definition nowadays
(because it is simpler).
M
M
RPMM
5%
10%
50%
M
10% Valley Definition FWHM Definition

141
dynode electron multiplier, in which the entire surface of the
multiplier is physically and electrically continuous. The interior surface of the electron
multiplier that is located near the entrance is
held at a highly negative potential (usually 1.2 to 3 kV); the exit end is referenced
to ground (0 V).

142
As each incoming ion collides with the multiplier surface,approximately two electrons
are ejected from the surface.
To the ejected electrons the remaining interior of the multiplier appears more positive
than the entrance does, so that they are attracted further into the multiplier where they
collide with the interior surface.
Each electron ejected by the second collision also results in the
ejection of two electrons, and this process continues down to the exit or last dynode
of the multiplier.
The total number of electrons ejected depends on the gain of the multiplier,
which is roughly a function of the total potential difference between the entrance
and exit to the multiplier surface. The gain can be adjusted daily during instrument
tune-up so that a standard quantity of a reference sample such as PFTBA will produce
approximately the same signal intensity. The total signal
amplification is approximately 2n, where n is the total number of collisions with
the multiplier surface. Most multipliers provide about a 105- to 106-fold increase
in signal—about 18–20 collisions. Electrons generated in the last collision with
the multiplier surface constitute the signal current output of the multiplier. This
current is sent to an external electronic signal amplification circuit and finally to
the data system.
1.4.2.

143
The most commonly used calibration
standard for routine GC/MS work is perfluorotri-n-butylamine [(CF3CF2CF2-
CF2)3N; PFTBA], which gives fragment ions over the range from m/z 30 – 600
. Prominent peaks at m/z 69, 219, and 502 in the spectrum of
this compound can be used to adjust settings for instrument variables
This compound exhibits peaks of at least moderate intensity over the entire
mass range normally used in GC/MS work

144
The largest
peak in the mass spectrum (100% relative intensity) is called the base peak
It is important to distinguish between the
terms ions and peaks in mass spectrometry. Ions are particles that have both mass
and charge, and they can fragment to form other ions. There can be large or small
numbers of ions, so that it is appropriate to speak of their relative abundance. On
the other hand, peaks in a mass spectrum correspond to localized maximum signals
produced by the detector and have only m/z values associated with them. These
signals
are either weak or strong (depending on the numbers of ions produced) and
therefore are best described as having intensity. The abundance of peaks implies
that there are many peaks, not that a given peak is big or little.

145
EI mass spectrum of methane is shown
Earth would contain 98.9% C atoms that were 12C and 1.1% that were 13C.
It may seem surprising that 14C is missing from this list, because it is undoubtedly
familiar to many readers as the basis for radioactive C dating in archaeology.
Although 14C is indeed a naturally occurring isotope of C, it undergoes continual
radioactive decay, which makes it unsuitable for determining elemental compositions

147
If this information is applied to methane, the MM of 12CH4 is calculated to be
16 u (12 u for the C and 1 u for each H), whereas that of 13CH4 is 17 u (13 u for the
C and 1 u for each H). Because ions are separated in mass spectrometry according
to their m/z values, the mass spectrum exhibits a peak for each of these ions. Indeed,
mass spectrometry offers one of the best ways to identify and quantify the presence
of different isotopes in a sample. The ratio of the intensities of the peaks at m/z 17
and 16 are directly related to the natural abundances of the two C isotopes (1.1% for
13C / 98.9% for 12C ¼ 1.1%).
The difference between the actual atomic mass of an
isotope (relative to 12C) and the nearest integral mass is called the mass defect,
which is denoted by the capital Greek letter DELTA.

148
Three of the elements (F, P, and I) occur without natural stable
isotopes. This means that these elements will contribute only one peak at a single
m/z value for each ion in which they occur.
The small amount of deuterium (2H) that occurs naturally (0.015%) is usually ignored
in the MS analysis of compounds
having M < 500 u because its contribution falls at or below the normal limits of
detection, which are often 0.1–0.5% of the base peak. This is not true for very large
molecules, however, because the 2H contribution for an ion containing even 100 H
atoms is 100 x0.015% = 1:5%.
For compounds containing only H, F, P, and I, or only one atom of an element
that has a naturally occurring isotope, isotopic abundance considerations are fairly
trivial.

MASS SPECTROMETRY(mass-spec) -2013 - P.ravisankar- WHAT ABOUT MASS SPECTROMETRY,BASIC PRINCIPLE,INSTRUMENTATION, ION SOURCES, MASS ANALYZERS,APPLICATIONS.

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