What is Mass Spectrometry?

Mass spectrometry is an analytical technique that involves the study

in the gas phase of ionized molecules with the aim of one or more of
the following:

Molecular weight determination

Structural characterization

Gas phase reactivity study

Qualitative and quantitative analysis of components in a

mixture.

Mass spectrometry consists basically of weighing ions in the gas

phase. The instrument used could be considered as a sophisticated
balance which determines with high precision the masses of individual
atoms and molecules. Depending on the samples chemical and physical
properties, different ionization techniques can be used. One of the
main factor in choosing which ionization technique to be used is
thermolability. For samples that are not themolabile and relatively
volatile, ionization such as Electron Impact and/or Chemical
Ionization can be effectively used. For samples that are thermolabile
such as peptides, proteins and other samples of biological interest,
soft ionization techniques are to be considered. Among the most used
soft ionization techniques are Electrospray (ESI) and Matrix Assisted
Laser Desorption (MALDI). The name given to a particular mass spec
technique is usually pointing to the ionization method being used.
Atomic and molecular masses are assigned relative to the mass of the
carbon isotope, 12C, whose atomic weight is defined as exactly 12. The
actual mass of 12C is 12 daltons, with one dalton is equal to 1.661
10-24 g. The mass of a molecule or an ion can be presented in daltons
(Da) or kilodaltons (kDa).

The Mass Spectrometer

Mass spectrometry uses an instrument called a mass spectrometer.
The main components of a mass spectrometer are:

Inlet system (LC, GC, Direct probe etc...)

Ion source (EI, CI, ESI, APCI, MALDI, etc...)

Mass analyzer (Quadrupole, TOF, Ion Trap, Magnetic Sector)

Detector (Electron Multiplier, Micro Channel Plates MCPs)

Samples can be introduced to the mass spectrometer directly via

solids probe, or in the case of mixtures, by the intermediary of
chromatography
device
(e.g.
Gas
chromatography,
Liquid
chromatography, Capillary electrophoresis, etc...). Once in the
source, sample molecules are subjected to ionization. Ions formed in
the source (molecular and fragment ions) acquire some kinetic energy
and leave the source. A calibrated analyzer then analyzes the passing
ions as a function of their mass to charge ratios. Different kind of
analyzer(s) can be used, Magnetic, Quadrulpole, Ion trap, Fourier
Transform, Time of Flight, etc...The ion beam exiting the analyzer
assembly is then detected and the signal is registered. Common
ionization method acronyms include:

EI=Electron Impact;

CI=Chemical Ionization;

SIMS=Secondary Ions Mass Spec;

FAB=Fast Atom Bombardment;

LDMS=Laser Desorption Mass Spec;

PDMS=Plasma Desorption Mass Spec;

TS=Thermospray;

AS=Aerospray;

ESMS=Electrospray Mass Spec.

Common mass analyzer acronyms include:

EB=Electrostatic-Magnetic;

IT=ion trap;

Q=Quadrupole;

TOF=Time of Flight.

Ionization Methods
Selection of the proper ionization method for the analysis of your
sample is extremely important. Although we can offer suggestions, it
is your responsibility to understand and select the method(s)
appropriate for your research compounds.

Electron Impact EI Ionization

Chemical Ionization CI

Negative Ion Chemical Ionization

Electrospray Ionization Techniques

Matrix Assisted Lazer Desorption (not offered in our facility,

but available elswhere on campus)

Atmospheric Pressure Chemical Ionization APCI

Electron Impact Ionization

M + e-(70eV) -----> M+. + 2eEI ionization method is suitable for non thermolabile compounds. The
volatility of the sample is required. Sample molecules in vapor state
are bombarded by fast moving electrons, conventionally 70 eV energy.
This results in ion formation. One electron from the highest orbital
energy is dislodged, and as a consequence molecular ions are formed.
Some of this molecular ions decompose and fragment ions are formed.
The fragmentation of a given ion is due to the excess of energy that
it requires within the ionization. Fragment ions can be odd electron
or even electron. Molecular ions formed in electron impact ionization
are odd electron ions. Odd electron fragment ions are formed by
direct cleavage(e.g. direct cleavage of a C-C bond). Even electron
fragment ions are often formed by rearrangement(e.g. proton
transfer). Sample can be introduced to the EI source via a gas
chromatography device, for example in the case of mixtures, or
directly via a solids probe device. The quantities needed for an
experiment is usually less than a microgram of material.
EI mass spectra, in most of cases, contain intense fragment ion peaks
and much less intense molecular ion peak. When the molecular ion peak
is not observed in the mass spectrum, chemical ionization can be used

in order to get molecular ion information. One helpful rule for

determining whether an ion is a molecular ion is the Nitrogen Rule.

Nitrogen rule: As indicated above, molecular ions formed in EI

ionization are odd electron ions. If their observed mass to
charge ratio is odd, the molecule under investigation contains
an odd number of nitrogen atoms. If that mass to charge ratio
is an even number, that molecule contains no or even Nitrogen
atoms.
Chemical Ionization
For organic chemists, Chemical Ionization (CI) is especially useful
technique when no molecular ion is observed in EI mass spectrum, and
also in the case of confirming the mass to charge ratio of the
molecular ion. Chemical ionization technique uses virtually the same
ion source device as in electron impact, except, CI uses tight ion
source, and reagent gas. Reagent gas (e.g. ammonia) is first
subjected to electron impact. Sample ions are formed by the
interaction of reagent gas ions and sample molecules. This phenomenon
is called ion-molecule reactions. Reagent gas molecules are present
in the ratio of about 100:1 with respect to sample molecules.
Positive ions and negative ions are formed in the CI process.
Depending on the setup of the instrument (source voltages, detector,
etc...) only positive ions or only negative ions are recorded.
In CI, ion molecule reactions occur between ionized reagent gas
molecules (G) and volatile analyte neutral molecules (M) to produce
analyte ions. Pseudo-molecular ion MH+ (positive ion mode) or [M-H](negative ion mode) are often observed. Unlike molecular ions
obtained in EI method, MH+ and [M-H]- detection occurs in high yield
and less fragment ions are observed.
Positive ion mode:
GH+ + M ------> MH+ + G
Negative ion mode:
[G-H]- + M ------> [M-H]- + G
These simple proton transfer reactions are true gas-phase Acid-Base
processes in the Bronsted-Lowrey sense.
A"tight" ion source
(pressure=0.1-2 torr) is used to maximize collisions which results in
increasing sensitivity. To take place these ion molecule reactions
must be exothermic. Proton transfer is one of the simple processes
observed in positive CI:
RH+ + M -----> MH+ + R
One of the decisive parameter in this reaction is the proton
affinity. For the reaction to occur, the proton affinity of the
molecule M must be higher that the one of the gas molecule. The main
reagent gases used in CI are: Ammonia, Methane, and Isobutane. The

predominant reactant ions formed are given in the mechanisms shown

below. Choice of reagent gas affects the extent of fragmentation of
the quasi-molecular ion.
Methane (positive ion chemical ionization):

CH4 + e -----> CH4+. + 2e ------> CH3+ + H.

CH4+. + CH4 -----> CH5+ +CH3.

CH4+. + CH4 -----> C2H5+ + H2 + H.

Isobutane (positive ion chemical ionization):

i-C4H10 + e -----> i-C4H10+. + 2e

i-C4H10+. + i-C4H10 ------> i-C4H9+ + C4H9 +H2

Ammonia (positive ion chemical ionization):

NH3 + e -----> NH3+. + 2e

NH3+. + NH3 ------> NH4+ + NH2.

NH4+ + NH3 --------->N2H7+

In methane positive ion mode chemical ionization the relevant sample

peaks observed are MH+, [M+CH5]+, and [M+C2H5]+; but mainly MH+.
This corresponds to the masses M+1, M+29, and M+41.
In isobutane positive ion mode chemical ionization the main peak
observed is MH+.
In ammonia positive ion mode chemical ionization the main peaks
observed are MH+, and [M+NH4]+. If more than one protonation site
is present, additional NH3 adducts might be seen corresponding to
[M+NH3+NH4]+. This corresponds to the masses M+1, M+18, and M+35.
In some cases, protonated dimers or other adducts might be seen; loss
of H2O followed by protonation or adduct ion formation is seen for
some classes of compounds. If the spectrum you observe does not seem
to show the proper adduct ions, or shows extensive fragmentation, be
wary when you try to interpret the results. There is an abundance of
data available in the literature discussing chemical ionization
mechanisms applicable to specific classes of compounds.
Two factors determine the choice of the reagant gas to be used:
1. Proton affinity PA
2. Energy transfer

NH3 (ammonia) is the most used reagent gas in CI because of the low
energy transfer of NH4+ compare to CH5+ for example. With NH3 as
reagent gas, usually MH+ and MNH4+ (17 mass units difference) are
observed.
Negative Ion Chemical Ionization
Three mechanisms can be underlined:
1. Electron capture reaction due to attainment of slow moving, low
energy "thermalized" electrons which may be transfered more
efficiently to sample molecules.
2. Electron transfer from ionized reagent gas (e.g. NH2- may
transfer an electron to a molecule having a greater electron
affinity than NH2).
3. Reagent gas ions participate in true CI reactions (e.g. proton
abstraction, according to relative acidities).
Molecular ions observed in negative ion chemical ionization mass
spectra are usually M- or [M-H]-.

Electrospray Ionization Method

Among the most used spray ionization techniques is Electrospray

Ionization (ESI). This technique continues to be the method of choice
for analyzing thermolabile chemicals. Its capabilities are well
documented. It uses an electrical stress between the ESI probe exit
(e.g. capillary) and the counter electrode, which is located few
millimeters from the probe. The process results in the generation of

highly charged droplets directly from the infused solution. Multiply

and/or singly charged analyte molecules desorbe from the sprayed
droplets and sampled through the rest of the mass spectrometer. ESI
has been distinguished for its ability to produce multiply charged
molecular ions from a large variety of polymers such as protein and
DNA fragments; it allows also sensitive detection of singly charged
low molecular weight polar species such as drugs and drug
metabolites. The formation of positive or negative ions (depending on
the sign of the applied electrical field) occurs in high yield. In
the positive ion mode protonated and/or alkali adduct analyte
molecules generally observed in the mass spectra. In the negative ion
mode operation peaks corresponding to deprotonated analyte molecules
are observed. ESI is described as a very "soft" ionization technique
where the surrounding bath gas has a moderating effect on the
internal and translational energies of desorbed ions.
Advantages of ESI:

Soft ionization process so intact molecular ions are

observed

ESI allows production of multiply charged ions. This

results in the ability of analyzing very high molecular
weight species using the most available mass analyzers
(e.g. quadrupoles).

ESI is an atmospheric pressure process. This makes it

easy to use and easy to interface with HPLC and CE
separation techniques.

Matrix Assisted Laser Desorption (MALDI)

Matrix Assisted Laser Desorption (MALDI) mass spectrometry technique

was introduced by Karas and Hillkamp in 1988 for the ionization of
peptides and proteins. Soon there after this technique was able to
analyze other type of biomolecules, such as oligosaccharides,
glycolipids, nucleotides, and synthetic polymers. In this technique,
samples are cocrystallized with a UV-absorbing substance called
matrix. For example for proteins, the matrix of choice is often
sinapinic acid. A 337 nm radiation from nitrogen laser is most
commonly used. The laser helps introducing energy into the molecular
system in such a way preventing thermal decomposition.
MALDI is often used with time-of-flights mass spectrometers ( TOF )
due to the pulsing nature of the technique, and the mass range
capability. Molecular weights up to few hundreds of daltons could be
measured. Comparison of MALDI and ESI ionization techniques was
attempted in the last few years. In my opinion these two techniques
are not competitive but complementary. Just to name a few, for high
molecular weight species, MALDI leads to the formation of singly
charged molecular ions while ESI allows the formation of multiply
charged molecular ions.
Practical considerations:

The final molar ratio sample/matrix is about or around

1/5000.

Final concentration of the sample is from 1 to 10

pmol/ul

Our experience with MALDI point to a dynamic range of

100 fmol/ul to few hundreds pmol/ul

MALDI is relatively robust ionization technique that

tolerates the use of salts and surfactants and buffers.
Although it is best to remove them for better
performance.

Peptide and Protein Standards for MALDI:

Angiotensin II (human)

Substance P (human)

Insulin (bovine)

Cytochrom c (equine)

RNase A (bovine)

Apo-Myoglobin (equine)

Trypsinogen (bovine)

MW: 1046.2

MW: 1347.7
MW: 5733.6
MW: 12,360.1

MW: 13,682.2
MW: 16,951.5
MW: 23,980.9

Atmospheric Pressure Chemical Ionization

APCI is a technique which creates ions at atmospheric pressure. A

sample solution flows through a heated tube where it is volatilized
in a mist and sprayed into a corona discharge with the aid of
nitrogen nebulization. Sample molecular are ionized by ion molecule
reactions from the ambiant corona discharge ions. Ions are produced
in the discharge and extracted into the mass spectrometer. APCI is
best suited to relatively polar, semi-volatile samples. An APCI mass
spectrum usually contained the quasi-molecular ion, [M-H]- or [M + H]
+.

Analysis of Ions
It is possible to use several different physical parameters to achive
mass seperation. Common types of mass analyzers are discussed
below. Each has advantages and disadvantages. In our facility we
currently have Quadrupole, Ion Trap, amd Time-of-Flight (TOF) mass
spectrometers.

Magnetic sector

Time of flight

Ion Trap

Quadrupole

Magnetic Sector Mass Spectrometer

The sector mass spectrometer was one of the most common types of mass
analyser and probably the most familiar to the everyday scientist. In
the 1950's, the first commercial mass spectrometers were sector
instruments. They consist of some combination of a large
electromagnetic, and some kind of electrostatic focussing device.
Different manufactures use differing geometries. Figure 1 shows a
schematic of a standard 'BE' geometry double focussing instrument.
The BE configuration is also called reverse geometry sector mass
spectrometer - that is, a dual sector instrument consisting of a
magnetic sector followed by an electrostatic sector.
Ions enter the instrument from the source (bottom left) where they
are initially focussed. They enter the magnetic sector through the
source slit where they are deflected according to the left-hand rule.
Higher-mass ions are deflected less than lower-mass ions. Scanning
the magnet enables ions of different masses to be focussed on the
monitor slit. At this stage, the ions have been separated only by
their masses. To obtain a spectrum of good resolution where all ions
with the same m/z appear coincident as one peak in the spectrum, ions
have to be filtered by their kinetic energies. After another stage of
focussing the ions enter the electrostatic sector where ions of the
same m/z have their energy distributions corrected for and are
focussed at the double focussing point on the detector slit.
Sector instruments had huge commercial successes in the 1950's and
1960's as they were the only practical way of obtaining highresolution data. In the last 20 years or so, with the decreasing
prices of FTMS and the development of high-resolution alternatives
(for example Q-Tof) sector instruments are in decline.
Time-of-Flight Mass Spectrometry (TOF-MS)
A time-of-flight mass spectrometer uses the differences in transit
time through a drift region to separate ions of different masses. It
operates in a pulsed mode so ions must be produced or extracted in
pulses. An electric field accelerates all ions into a field-free
drift region with a kinetic energy of qV, where q is the ion charge
and V is the applied voltage. Since the ion kinetic energy is 0.5mv2,
lighter ions have a higher velocity than heavier ions and reach the
detector at the end of the drift region sooner.

Theory:

K.E. = qV

1/2 mv2 = qV

v = (2qV/m)1/2

The transit time (t) through the drift tube is L/v where L is
the length of the drift tube

t=L / (2V/m/q)1/2

Schematic of a linear TOF-MS

This schematic shows ablation of ions from a solid sample with a

pulsed laser. The reflectron is a series of rings or grids that act
as an ion mirror. This mirror compensates for the spread in kinetic
energies of the ions as they enter the drift region and improves the
resolution of the instrument. The output of an ion detector is
displayed on an oscilloscope as a function of time to produce the
mass spectrum.

Ion Trap

Ions created by electron impact (EI), electrospray (ESI), or matrixassisted laser desorption (MALDI) ionization are focused using an
electrostatic lensing system into the ion trap. An electrostatic ion
gate pulses open (-V) and closed (+V) to inject ions into the ion
trap. The pulsing of the ion gate differentiates ion traps from
"beam" instruments such as quadrupoles where ions continually enter
the mass analyzer. The time during which ions are allowed into the
trap, termed the "ionization duration", is set to maximize signal
while minimizing space-charge effects. Space-charge results from too
many ions in the trap that cause a distortion of the electrical
fields leading to an overall reduction in performance. The ion trap
is typically filled with helium to a pressure of about 1 mtorr.
Collisions with helium dampens the kinetic energy of the ions and
serve to quickly contract trajectories toward the center of the ion
trap, enabling trapping of injected ions. Trapped ions are further
focused toward the center of the trap through the use of an
oscillating potential, called the fundamental rf , applied to the
ring electrode. An ion will be stably trapped depending upon the
values for the mass and charge of the ion, the size of the ion trap
(r), the oscillating frequency of the fundamental rf ( w), and the
amplitude of the voltage on the ring electrode ( V). The dependence
of ion motion on these parameters is described by the dimensionless
parameter qz, qz = 4eV/mr2w2

Quadrupole

A quadrupole mass filter consists of four parallel metal rods

arranged as in the figure below. Two opposite rods have an applied
potential of (U+Vcos(wt)) and the other two rods have a potential of
-(U+Vcos(wt)), where U is a dc voltage and Vcos (wt) is an ac
voltage. The applied voltages affect the trajectory of ions traveling
down the flight path centered between the four rods. For given dc and
ac voltages, only ions of a certain mass-to-charge ratio pass through
the quadrupole filter and all other ions are thrown out of their
original path. A mass spectrum is obtained by monitoring the ions
passing through the quadrupole filter as the voltages on the rods are
varied. There are two methods: varying w and holding U and V
constant, or varying U and V (U/V) fixed for a constant w.

Tandem Mass Spectrometry:

Tandem Mass Spectrometry, usually referred to as MS/MS, involves the
use of 2 or more mass analyzers. It is often used to analyze
individual components in a mixture. This technique adds specificity
to a given analysis. Although tandem mass spectrometry can be
referred to MS/MS, MS/MS/MS, etc..., in this presentation I am going
to describe only MS/MS.
The basic idea of MS/MS is a selection of a m/z of a given ion formed
in the ion source, and subject this ion to fragmentation, usually by
collision with inert gas (eg. Argon). The product ions are then
detected. This is a powerful way of confirming the identity of

certain compounds and determining the structure of unknown species.

So MS/MS is a process that involves 3 steps: ionization,mass
selection,mass analysis.
MS/MS could be performed on instruments such as triple quadrupole
(QQQ), ion trap, time of flight, fourier transform, etc... The triple
quadrupole is the most frequently used mass spectrometer for MS/MS,
perhaps because of the cost and ease of use among other factors.

Separation Methods for Coupling with Mass Spec

GC-MS: Sample mixtures are directly vaporized and enter bonded

fused silica columns. Components of the mixture are separated
based on their affinity difference with the bonded phase.
Separated compounds exit the column and enter the vacuum system
of the mass spectrometer. Sample molecules are ionized (EI, or
CI), and accelerated into a precalibrated mass analyzer (e.g.
Q, Ion Trap, TOF, FTMS etc...). Retention times, molecular
masses, and fragmentation patterns are recorded. One of the
most important considirations of GC/MS is that the sample(s)
must be non thermolable meaning thermally stable.

LC-MS: For compounds that are thermally unstable, LC/MS method

is considered. The separation is based on the diffrence in
affinity of samples with stationary phase and the mobile phase.
e.g. hydrophobicity in case of RP chromatography.

CZE-MS: This method is based on diffrences of electrophoretic

mobility of samples when the fused silica column is under a
potential difference between injection side and detector side.

CIEF-MS: This is a variant of CZE. It is based on diffrerences

in isoelectric points of analytes.