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Mass Spectrometry





Dr Nizam M. El-Ashgar
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Mass Spectrometry


Mass spectrum is obtained by converting components of
a sample into rapidly moving gaseous ions and resolving
them on the basis of their mass-to-charge ratios.

Most generally applicable of analytical tools since
capable of qualitative and quantitative information about
both atomic and molecular composition of inorganic and
organic compounds.

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Molecular Mass Spectrometry
Provides information about:
1- The elemental composition of samples of
matter
2- The structures of inorganic, organic and
biological molecules.
The qualitative and quantitative composition
of complex mixture
The structure and composition of solid
surfaces
Isotope ratios of atoms in samples.

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Acuracy of MWt Mesurments
For large samples such as biomolecules, molecular masses can be
measured to within an accuracy of 0.01% of the total molecular
mass of the sample.
Example: an error within a 4 Daltons (Da) or atomic mass units
(amu) for a sample of 40,000 Da. This is sufficient to allow minor
mass changes to be detected, e.g. the substitution of one amino
acid for another, or a post-translational modification.

For small organic molecules the molecular mass can be measured
to within an accuracy of 5 ppm or less, which is often sufficient to
confirm the molecular formula of a compound, and is also a
standard requirement for publication in a chemical journal.
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Where are mass spectrometers used?
Mass spectrometers are used in industry and academia for both
routine and research purposes. The following list is just a brief
summary of the major mass spectrometric applications:
Biotechnology: the analysis of proteins, peptides, oligonucleotides
Pharmaceutical: drug discovery, combinatorial chemistry,
pharmacokinetics, drug metabolism
Clinical:, haemoglobin analysis, drug testing
Environmental: water quality, food contamination
Geological: oil composition

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Mass Spectra
When the electron beam ionizes the molecule, the species that
is formed is called a radical cation, and symbolized as M
+
.
The radical cation M
+
is called the molecular ion or parent ion.
The mass of M
+
represents the molecular weight of M.
Because M is unstable, it decomposes to form fragments of
radicals and cations that have a lower molecular weight than
M
+
.
The mass spectrometer analyzes the masses of cations.
A mass spectrum is a plot of the amount of each cation (its
relative abundance) versus its mass to charge ratio (m/z, where
m is mass, and z is charge).
Since z is almost always +1, m/z actually measures the mass
(m) of the individual ions.
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Mass Spectrometry
The tallest peak in the mass spectrum is called the base peak.
The base peak is also the M peak, although this may not always
be the case.
Though most C atoms have an atomic mass of 12, 1.1% have a
mass of 13. Thus,
13
CH
4
is responsible for the peak at m/z = 17.
This is called the M + 1 peak.
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Since the molecular ion is unstable, it fragments into
other cations and radical cations containing one, two,
three, or four fewer hydrogen atoms than methane itself.
Thus, the peaks at m/z 15, 14, 13 and 12 are due to these
lower molecular weight fragments.
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Molecular Mass Spectra (hexane)
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Explanation
The collisions between energetic electrons and analyte
molecules (enough E) lead to excitation.
Relaxation leads to fragmentation to lower masses ions
Attraction of positive ions through the slit of a mass
spectrometer and sorted according to m/z ratios and
appear in the Mass spectrum.
Mass spectrum is a plot of relative intensity versus m/z
Base peak has the value of 100 (arbitrarily)
The remaining computed as % of the base-peak height
Modern MS programmed base-peak and normalize the
remaining peaks relative to that peak
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Mass Spectrometer
All Instruments Have:
1. Sample Inlet
2. Ion Source
3. Mass Analyzer
4. Detector
5. Data System
http://www.asms.org
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Ion sources
Starting point: Formation of gaseous analyte Ions.
Methods of ion formation: Two major categories:
1- Gas-phase sources
-The sample is first vaporized and then ionized.
-Restricted to thermally stable compounds of BP < 500
0
C.
-Limited to Compounds of MWts <10
3
dalton .
2- Desorption sources
The sample in a solid or liquid state is converted directly
into gaseous ions (not require volatilization of analyte
molecules)
Applicable to nonvolatile and thermally unstable samples
Applicable to analytes having of 10
5
dalton or larger.

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Basic Type Name Ionizing agent
Electron Impact (E I) Energetic electrons
Chemical Ionization (CI) Reagent gaseous ions
Gas Phase Field ionization (FI) High-potential electrode
Desorption
Field desorption (FD) High-potential electrode
Electrospray ionization (ESI) High electric Field
Matrix-assisted desorption/ionization
(MALDI)
Laser beam
Plasma desorption (PD)
Fission fragments from
252
Cf
Fast atom bombardment (FAB) Energetic atomic beam
Secondary ion mass spectrometry (SIMS) Energetic beam of ions
Thermospray ionization (TI) High temperature
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Hard and Soft Sources (another classification)
Hard Sources:
Impart sufficient E to analyte molecules (become of high excited E
state).
Relaxation involves rupture of bonds producing fragment ions with
m/z < than that of molecular ion.
Provides useful information about kinds of functional groups and
structure information.
Soft Sources:
Causes little fragmentation.
Mainly the spectrum consists of the molecular ion peak and few
peaks
Supplies accurate information about MWt of analyte.
Both are useful for analysis
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The Electron-Impact Sources (EI)

The sample is brought to a temperature high enough to produce a molecular
vapor, which is then ionized by bombarding the resulting molecules with a
beam of energetic electrons.
Positive ions forced by small potential difference through accelerator plates
to mass analyzer.

M + e
-
M
.+
+ 2e
-
where, M = analyte molecule,
M
.+
= molecular ion.
Relaxation then usually takes place by extensive fragmentation, giving a
large number of positive ions of various masses that are less than that of the
molecular ion. These lower mass ions are called daughter ions.

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Gaseous sample bombarded with beam of energetic electrons.
Electrons produced at heated W or Rh wire and accelerated to energy of
about 70 eV. Typically one in every million molecules undergoes ionization.

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Electron Impact (EI)
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The positive ions produced are attracted through the slit
in the first accelerating plate by a small potential diff (5
V).
With magnetic sector instrument high potentials (10
3
to
10
4
) are applied to the accelerator plates)
KE = qV = zeV
KE of an ion is independent of its mass and depends
only upon its charge and accelerating potential
Velocity of an ion depends on its mass
KE = 1/2mv
2
or v = (KE/m)
1/2

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Electron Impact
Advantages
Well-Established
Fragmentation Libraries
Insoluble Samples
Interface to GC
Non-Polar Samples
They are convenient and produce high ion currents.
Extensive fragmentation can lead to unambiguous identification of analytes
Disadvantages
Parent Identification
Need Volatile Sample
Need Thermal Stability
No Interface to LC
Low Mass Compounds (<1000 amu)
Solids Probe Requires Skilled Operator
The need to volatilize the sample limits this method since it excludes
analysis of thermally unstable compounds.
Excessive fragmentation can lead to the disappearance of the molecular ion
peak therefore preventing the molecular mass of the analyte to be
determined.


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Electron-Impact Spectra
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EI-MS of Methylene Chloride (top) and 1-Pentanol (bottom)
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Types of MS Peaks:

Molecular (or Parent) Ion ion having same mass
as the analyte
Daughter Ion ion having lower mass
Base Peak biggest peak

Size of peaks depends on relative natural
abundance of isotopes
The base peak in electron-impact spectra arise from
fragments rather than from molecular ion.
The molecular ion peak provides the MWt of the
unknown.
In EI certain molecules yield no molecular ion peak.

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Isotope Peaks
Peaks occur at masses that are greater than that of the molecular ion are
attributable to ions containing isotopes.
Examples:
12
C
1
H
2
35
Cl
2
: m = 84
13
C
1
H
2
35
Cl
2
: m = 85
12
C
1
H
2
35
Cl
37
Cl: m = 86
13
C
1
H
2
35
Cl
37
Cl: m = 87
12
C
1
H
2
37
Cl
2
: m = 88
The size of various peaks depends upon the relative natural abundance of the
isotopes.
Note: F, P, I, Na occur only as single isotopes.
Isotope peaks some times provide useful means for determining the formula for
compound.

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Collision product peaks
Ion/molecule collisions, can produce peaks at higher
mass numbers than that of the molecular ion.
At ordinary sample pressures, however the only
important reaction of this type: transferring of H atom to
the ion to give a protonated molecular ion, enhanced (M
+1 )
+
peak results (second order reaction).
The amount of product depends strongly upon the
reactant concentration.
The height of an (M+1)
+
peak due to this reaction
increases with increase in sample pressure than do the
heights of other peaks (detected).
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Advantages of EI-MS
Convenient to use
Produce high ion currents (gives good sensitivities)
Often unambiguous identification of compounds possible
(extensive fragmentations)

Disadvantages of EI-MS
Extensive fragmentation may not leave parent ion
(Molecular peak ion).
Sample must be volatilized (thermal degradation) of some
analytes before ionization to be occur.
Only applicable to samples with molecular weights < 10
3
amu
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Chemical ionization sources and spectra
Second most common process for ms
In modern MS, EI ionization and chemical ionization are interchangeable.
Gaseous atoms are ionized by collision with ions produced by electron
bombardment of an excess of a reagent gas.
Positive ions usually used.
negative ion used with analyte contain very electronegative atoms.
Experimentally:
Modification of the electron beam ionization area of EI by adding
vacuum pump capacity and reducing width of the slit to the mass
analyzer so P reduced to 1 torr (Ionization area) and 10
-5
torr in the
analyzer.
Gaseous regent introduced in the ionization region reagent:sample =
10
3
-10
4
. So electron beam reacts exclusively with reagent
molecules.
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Common Reagents
Methane
Reacts with high E electrons to give several ions (CH
4
+
and CH
3
+
(90 %),
CH
2
+
).
These ions react rapidly with additional methane molecules:
CH
4
+
+ CH
4
CH
5
+
+ CH
3
CH
3
+
+ CH
4
C
2
H
5
+
+ H
2
Reactive collisions between sample molecules MH and CH
5
+
or C
2
H
5
+
take
place: (proton and hydride transfer).
CH
5
+
+ MH MH
2
+
+ CH
4
(proton transfer) gives (M +1)
+
ion peak
C
2
H
5
+
+ MH MH
2
+
+ C
2
H
4
(proton transfer) gives (M +1)
+
ion peak
C
2
H
5
+
+ MH M
+
+ C
2
H
6
(hydride transfer) gives (M -1)
+
ion peak
C
2
H
5
+
+ M M- C
2
H
5
+
gives (M + 29)
+
ion peak
Other reagents: propane, isobutane and ammonia that give different spectra
with a given analyte.

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EI hard
source
CI Soft
source
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Field ionization Sources
Ions are formed under large electric field (10
8
V/cm).
The field produced by applying high voltages (10 to 20KV) to emitters
having diameter < 1 m.
Example: W fine wire (~ 10 m diameter) emitter of many hundreds of
carbon microtips projecting from the surface tungsten wire.
FI are mounted 0.5-2 mm
from the cathode (often serves as slit).
Gaseous sample is allowed to
diffuse into the high-field area
around the microtips of the anode.
EF is concentrated at the emitter tips
and ionization occurs via a quantum mechanical tunneling mechanism in which
electrons from the analyte are extracted by microtips of the anode.
Little vib. or rot energy is imparted to the analyte thus little fragmentation
occurs.

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FI Source
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MS of Glutamic Acid
Electron Impact
Field Ionization
Field Desorption
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Desorption sources
Previous methods: The ionizing agents act on gaseous samples.
Such methods are not applicable to nonvolatile or thermally unstable
samples.
Desorption ionization methods: are develop to deal with this type of
samples. (thermally unstable biochemical species or that of 100, 000 Da).
The method is: dispenses the volatilization followed by ionization of the
gaseous analyte molecules.
Instead: E in various forms is introduced into solid or liquid sample large
molecules to overcome intermolecular forces to cause direct formation
of gaseous ions.
provide gentle ionization. e.g. carbohydrates, peptides, nucleic
acids, organic salts & organometallics.

Simple spectra consist of only the molecular ion or the protonated molecular
ions.
Exact mechanism is not understood.
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Field Desorption Sources
Multitipped emitter similar to FI source is used.
The electrode is mounted on a probe that can removed from the
sample compartment and coated with a solution of the sample.
The probe is reinserted into the sample compartment.
Ionization takes place by the application of a high potential to this
electrode.
Some samples needed heating the emitter by passing a current
through the wire.
Thermal degradation may occur before ionization is complete.
Example of spectra (glutamic acid previously).
Spectrum is simpler than that of CI or FI consists of only the
protonated molecular ion peak at mass 148 and isotope peak at
mass 149.
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Matrix-Assisted Laser Desorption/Ionization (MALDI)
MALDI: is a new ionization method: provides accurate MWt of polar
biopolymers (MWt few thousands to several hundred thousands da).
First technique (German group): an aqeous/alcohol solution of
sample was mixed with large excess of a radiation-absorbing matrix
material.
The resulting solution was evaporated on the surface of a metallic
probe that was used for introduction of the sample into mass
spectrometer.
The solid mixture exposed to a pulsed laser beam so analyte ions
sublime.
Entire spectrum recording between the period of laser pulses.
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hv
Laser
1. Sample is mixed with matrix (X)
and dried on plate.
2. Laser flash ionizes matrix
molecules.
3. Sample molecules (M) are
ionized by proton transfer:
XH
+
+ M MH
+
+ X.

MH
+
MALDI: Matrix Assisted Laser Desorption Ionization
+/- 20 kV
Grid (0 V)
Sample plate
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Requirement for MALDI:
Matrix compound must absorb the laser radiation strongly.
Soluble in sample solvent and present in large excess in the solid
matrix deposited on the probe.
The analyte should not absorb laser to prevent fragmentation.
Only few compounds are suitable as matrices for biopolymers
(Table 20-4).
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6. MALDI

matrix has strong A at laser
low mass, so can sublime
can measure M
r
of proteins and biomolecules

500 fmol of beta-
D-galactosidase
mixed with nicotinic
acid on a silver
surface using Nd-
YAG 266 nm
excitation
RMM 117130
Fragments are
cluster ions [nM+H]
+

and multiply-
charged ions
[nM+zH]
z+
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Electrospray ionization
ESI/MS (1984) now it is an important technique for analyzing
biomolecules (polypeptides, proteins and oligonucleotides having
MWt 0f 100,000 Da or more). Also used to identify inorganic species
and synthetic polymers).
Takes place under atmospheric pressures and temperatures.
A solution of the sample is pumped through a stainless steel
capillary needle at a rate of few microliters per minute.
The needle is maintained at several kilovolts with respect to a
cylindrical electrode surrounds the needle.
The resulting charged spray of fine droplets then passes through a
dissolving capillary .
Solvent evaporate and analyte charged and charge density
increases and ions desorbed into the ambient gas.
Little fragmentation of large thermally fragile biomolecules occurs.
The ions formed are multiply charged (small m/z) detected by
quadrupole instruments (range of 1500 or less).
Is ready adapted to direct sample introduction from HPLC and
electrophoresis columns.

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ESI ms in 50%
CH
3
CN/46%
H
2
O/4%
acetic acid
with source at
100
o
C.

proteinase K
29 kDa
thermolysin
34 kDa
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Fast Atom Bombardment FAB
FAB is a major role in the production of ions for ms studies of polar
high MWt. species.
Samples in a condensed state often in a glycerol solution matrix are
ionized by bombardment with energetic (several KeV) xenon or
argon atoms.
Both +ve and ve analyte ions are sputtered from the surface of the
sample in a desorption process.
Rapid sample heating by this process reduces sample
fragmentation.
A beam of fast atoms is obtained by passing accelerated Ar or Xe
ions from an ion source or gun through a chamber containing Ar or
Xe atoms at P of about 10
-5
torr.
The high velocity ions undergo exchange reactions with the atoms
without loss of translational energy and a beam of energetic atoms
is formed.
The lower E ions from the exchange are removed by an electrostatic
deflector.
FAB of organic or biochemical compounds usually produces
significant amounts of molecular ions (and ion fragments) even for
high MWt and thermally unstable samples (over 10,000 da) and
detailed structure information obtained for samples of (3000 da).


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FAB sputtering/desorption technique.

Xe/Ar atoms
Atoms/ions
penetrate several
layers, distribute
k.e.
No fragment
n
Sample dissolved in low-volatility, viscous solvent and placed on
insertion probe. Bombard with atoms or ions 4-10 keV energy.
up to 9600 amu
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Sample monolayer
sputtered in few s.
Nonvolatile
glycerol allows
surface layer to be
renewed.

Ar G (G
n
+H)
+
(M+H)
+
sample M (M-H)
-
(M+G+H)
+
FAB
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Mass Spectrometer
Several Types. Only two will be described:
Quadrupole spectrometer and the time-of-flight spectrometer.
General description of instrument components:

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Inlet System:
To introduce a very small amount of sample (mol or less) into the mass
spectrometer that converted to gaseous ions. ( a mean for volatilizing solid
or liquid samples is presents).
Ion sources:
Convert the components of a sample into ions.
(generally the inlet system and the ion source are combined into a single
component). The output is a stream of +ve or ve ions are accelerated into
the mass analyzer.
Mass analyzer
analogous to grating in an optical spectrometer.
Dispersion is based upon the mass/charge ratios of the analyte ions rather than
upon the wavelength of photons. (Different categories of MS according to
mass analyzer).
Detectors:
Convert the beam of ions into an electrical signal that can then be
processed, stored in the memory of a computer and displayed or
recorded in a variety ways.
Vacuum System: To create low pressure (10
-4
to 10
-8
torr) in all the
instrument components except the signal processor and readout. To
prevent interaction of components with atmosphere so destroyed.
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Sample Inlet System
For permitting introduction of a representative sample into the ion
source with minimal loss of vacuum. Various types of inlets
equipped to accommodate different samples: (Batch inlets, direct
probe inlets, chromatographic inlets and capillary electrophoretic
inlets).
Batch Inlet Systems:
Classical and simplest type.
Sample is volatilized externally and then allowed to leak into evacuated
ionization region.
The figure shown is for a one that applicable to gaseous and liquid
samples having PB up to 500
0
C.
Gaseous samples: A small measured volume of a gas is trapped
between the two valves and then expanded into reservoir flask.
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For liquids a small quantity of sample is introduced into a reservoir usually
with a micrometer syringe.
In either case vacuum system is used to achieve sample P 10
-4
10
-5
torr.
For samples with boiling points > 150
0
C T must be maintained at elevated T
by oven and heating tapes of maximum T of about 350
0
C.
Sample is now in the gas phase is leaked into the ionization area of the
spectrometer via a metal or glass diaphragm containing one or more
pinholes.
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MS Direct Sample Introduction
External Sample Introduction System
Direct Sample Probe
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The Direct Probe Inlet.
Solids and non volatile liquids can be introduced into the ionization region
by means of a sample holder probe which is inserted through a vacuum
lock.
The lock system:
Designed to limit V of air pumped after probe insertion.
Probes also used for limited quantity samples (few nanograms)(less wasted
than batch system).
Probe: Sample is held on the surface of a glass or Al capillary tube, a fine wire
or a small cup.
The probe is positioned within a few mm of the Ionization source and the slit
leading to spectrometer.
Vacuum used to maintain thermally unstable compounds for spectrum
measurements before major decomposition occurs. And to elevate
nonvolatile conc. in the ionization area (carbohydrates, steroids, metal-
organic species and low molecular weight polymeric substances).
Partial pressure attained is at least of 10
-8
torr before onset decomposition.
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Chromatographic and capillary electrophoretic inlet System.
MS are often coupled with GC and HPLC or capillary
electrophoresis columns.
Separation and determination of the components of complex
mixture is obtained.
Specialized inlet systems is needed.
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Mass Analyzers
Several devices are available for separating ions with different m/z ratios.
Mass analyzer should be:
- Capable of distinguishing between minute mass differences.
- Allows passage of sufficient number of ions to yield measurable currents.
Resolution of mass spectrometer: R
R = m/Am
Where, m : mass of the first peak (or mean mass of the two peaks)
Am: mass difference between two adjacent peaks
Two peaks are considered to be separated if height of valley between them s
10% of their height.
R of 4000 would resolve peaks occurring at m/z values of 400.0 and 400.1 (or
40.00 and 40.01)




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R needed in MS depends upon its application.
Example 1: same nominal mass ions: C
2
H
4
+
, CH
2
N
+
, N
2
+ and CO
+

(All ions of nominal mass 28 Da).
Exact masses: 28.0313, 28.0187, 28.0061 and 27.9949 Da respectively.
These requires an instrument with a resolution of several thousands.
Example 2: Low MWt ions with a unit mass difference or more: NH
3
+
(m = 17)
and CH
4
+
(m = 16) R instrument of 50 or less is sufficient.

Commercial MS are available with R range of 500-500,000.
Example:
What R is needed to separate C
2
H
4
+
and CH
2
N
+
, ions?
Am = 28.0313 - 28.0187 = 0.0126
R =m/ Am = 28.025/0.0126 = 2.22x10
3
Where 28.025 is the mean mass for the two species
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Magnetic Sector Analyzers
Employ a permanent magnet or an electromagnet.
Cause beam from the ion source to travel in a circular path most
commonly of (180, 90 or 60 deg)
Schematic of a 90-deg magnetic sector spectrometer

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Operation:
Ions formed by Electron impact are accelerated through slit B into the metal
analyzer tube internal P ~ 10
-7
torr.
Ions of different mass can be scanned across the exit slit by varying the
field strength of the magnet or the accelerating potential between slits
A and B.
Ions passing through the exit slit fall on a collector electrode, ion current
resulted that amplified and recorded.
Translation or KE of an ion of mass m bearing z exiting slit B is given by:
KE = ZeV = mv
2

Where V is voltage between A and B
V is velocity of the ion after acceleration
e is the electronic charge = 1.60x10
-19
C
Note: all ions having the same number of charges are assumed to have the
same KE after accelerating regardless of their mass. (approximately true).
All ions leaving the slit have approximately same KE, the heavier ions must
travel at lower velocities.
The path in sector by ions of a given m/z represents a balance between two
forces acting upon them.

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The magnetic force F
M:
F
M
=BzeV
Where B is MF strength
The balancing centripetal force:
F
c
= mv
2
/r
Where r is the radius of curvature of the magnetic sector.
For ion to traverse the circular path to the collector,
F
M
and F
C
must be equal

F
M
=BzeV = F
c
= mv
2
/r
v = Bzer/m
Substituting the previous equation in ZeV = mv
2

m/z = B
2
r
2
e/2V
Mass Spectra:
Varying one of three variables (B, V or r) while holding the other two constants.
Modern MS ions are sorted by holding V and r constant while varying the
current in the magnet and thus B.
In sector MS (using photographic recording) B and V are constants, r is the
variable.


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What accelerating potential will be required to direct a singly charged water molecule
through the exit slit of a magnetic mass spectrometer if B = 0.240 T and r of curvature
of the ion through the magnetic field is 12.7 cm?
SI units:
ez = 1.60x10
-19
Cx 1 r = 0.127 m






m = 2.99x10
-26
kg
B = 0.240 T = 0.240 W/m
2
m/z = B
2
r
2
e/2V or V = B
2
r
2
ez/2m
= [0.240 W/m
2
]
2
[0.127m]
2
[1.60x10
-19
C]
2x2.99x10
-26
kg
= 2.49 x10
3
W
2
C/m
2
kg or volts

(In SI base units, the dimensions of the weber are kg.m
2
/S
2
.A . In derived units, they
are volt-seconds V.s , or joules per amp J/A.
. maxwells
8
10 =
2
m T 1 The weber is a large unit, equal to

g
kg
x
mol O H x
mol O H g
m
3
2
23
2
10
/ 10 02 . 6
/ 02 . 18

+
+
=
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Double focusing spectrometers
Previous are single-focusing spectrometers.
Limitation: Low precision because of:
1- directional distribution of ions
2- energy distribution of ions
Same m/z ratio but with small diverging directional distribution are
focused so limiting the resolution of magnetic sector instruments
(Rs2000).
This because of the translational E distribution of ions leaving a source
(Boltzmann dist.) arises from energies of original molecules and
source field inhomogeneities.
Spread of KE causes a broadening of the beam reaching the
transducer and a loss of resolution.

4:17 AM 5/4/2013 63
Double Focusing Spectrometers:
Correction for both the directional and E distribution of ions.
Both directional and E aberrations of a population of ions are simultaneously
minimized by use of carefully selected combinations of electrostatic and
magnetic fields.

4:17 AM 5/4/2013 64
The ion beam is first passed through an electrostatic analyzer (ESA).
Consists of two smooth curved metallic plates across which a dc potential is
applied which limiting the KE of the ions reaching the magnetic sector to a
closely defined range.
Ions with E greater than average strike the upper side of ESA slit and lost to
the ground.
Ions with E less than average strike the lower side of the ESA slit and are
thus removed.
Directional focusing occurs along the focal plane (d).
Energy focusing takes place along the plane e.
Only ions of one m/z are double focused at the intersection of d and e for
any given V and B.
The collector slit is located at this locus of double focus.
4:17 AM 5/4/2013 65
Quadrupole Mass Spectrometer
Comparing with magnetic sector instrument it is:
Less expensive and more rugged.
More compact
Found in commercial bench top ms
Low scan times (<100 ms) which is useful for
chromatography.
Most common mass analyzers in use today.
Quadrupole mass analyzer is responsible for filtering the samples
ions.
Consists of four parallel metal rods.
Each rod pair is connected together electrically.
A radio frequency voltage is applied between one pair of rods then
the other.

4:17 AM 5/4/2013 66
Ions travel down the
quadrupole in between the rods
Electric field separates ions
Ions are subjected to complex
forces
Only Ions of a particular m/z
reaches the detector
Advantages
Inexpensive
Easily Interfaced to Many
Ionization Methods
Disadvantages
Low Resolution (< 4000)
Low Accuracy (>100 ppm)
MS/MS requires multiple
analyzers
Low Mass Range (< 4000)
Slow Scanning
4:17 AM 5/4/2013 67
Time-of-Flight MS (TOF)
The +ve ions are produced periodically by bombardment of the
sample with brief pulses of electrons, secondary ions or laser
generated photons.
The ions are then accelerated into a field-free drift tube by an
electric field pulse of 10
3
10
4
V
Separation of ions on the basis of mass occurs during the
transition of the ions to the detector located at the end of the tube.
All ion have same KE but their velocities are inversely proportional
to their masses
Lighter particles arrived earlier.
Typical flight times are 1 to 30 s.

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Time-of-Flight MS (TOF)

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4:17 AM 5/4/2013 70
Advantages
Simplicity and ruggedness.
Ease of accessibility of the ion source.
Extremely High Mass Range (>1 MDa)
Fast Scanning
Disadvantages
Low Resolution (4000)
Low sensitivity
Low Accuracy (>200ppm)
MS/MS not possible

4:17 AM 5/4/2013 71
Ion Trap Analyzers
Gaseous anions or cations can be formed and confined for extended
periods by electric and/or magnetic fields.
Several types, A simpler type of ion trap that used for GC/MS
Now used to obtain mass spectra of a variety of analytes.
4:17 AM 5/4/2013 72
4:17 AM 5/4/2013 73
It consists of a central doughnut-shaped ring electrode and a pair of endcap
electrodes.
A variable RF voltage is applied to the ring electrode while the two end-cap
electrodes are grounded.
Ions of appropriate m/z value circulate in a stable orbit within the cavity
surrounded the ring.
By increasing RF voltage the orbit of heavier ions become stabilized , while
lighter become destabilized causing them to collide with the wall of the ring
electrode.
By RF voltage scanning the trapped ions destabilized and leave the ring
electrode cavity via openings in the lower end cap then emitted to a
transducer.
Rugged compact and less costly than sector or quadrupole instruments.
Capable of resolving ions that differ in mass by unit in the mass range of
500-1000 Da


4:17 AM 5/4/2013 74
Operation of an Ion Trap MS
4:17 AM 5/4/2013 75
Fourier Transform (FT) instruments
FTMS provide:
improved signal to noise ratios
Greater speed.
Higher sensitivity.
Higher resolution.
The heart of FTMS is an ion trap within which ions can circulate in a
well-defined orbits for extended periods.

4:17 AM 5/4/2013 76
The Ion Cyclotron Resonance (ICR)
Gaseous ion drifts into or formed in a strong MF.
The motion becomes circular in a plane that is to the direction of
the field.
The angular frequency of this motion is called cyclotron frequency
e
c
.
e
c
= v/r = zeB/m
In fixed field the e
c
depends only upon the inverse of the m/z value.
Increases the velocity of an ion will be accompanied by increase of
rotation of the ion.
This circulated trapped ion in the MF is capable of absorbing E from
an ac EF.
So the EF frequency matches the e
c.
The absorbed E increases v of the ion and r of travel without
disturbing e
c

4:17 AM 5/4/2013 77
MS Fourier Transform Analyzer
Ion Cyclotron Resonance
Magnetic Field
4:17 AM 5/4/2013 78
Inner solid line represents the original path to the ion.
Dashed line shows spiral path when switch is moved briefly to
position 1.
Outer solid line is new circular path when switch again opened.
For ensemble ions of the same m/z ratio between the two plates;
when ac signal is applied the cyclotron resonance frequency sets all
the particles into coherent motion that is in phase with the field.
Ions of different m/z ratios (different e
c
) are unaffected by the ac
field.
Measurement of the ICR signal:
Coherent circular motion of resonant ions creates image current
observed current after termination of the frequency sweep signal
(from position 1 to 2).
The current decreases exponentially with time.
It is a capacitor current induced by circular movement of a packet of
ions with the same m/z ratios.
4:17 AM 5/4/2013 79
Example:
+ve ions approaches the upper plate electrons attracted from circuit
common to this plate causing a momentary current.
This current reversed at the other plate as ions reach the other plate.
An ac current produced depends on number of ions in the packet
Frequency of ac current is characteristic of m/z value of the ions in the
packet.
This current measures conc. of ions.
The induced image current decays with time (few tenth of s to several
s) by losing energy with collisions between ions (ions reach the
thermal equilibrium) (time domain signal).
4:17 AM 5/4/2013 80
Diagram of the cell used in pulsed ICR & in FT-MS
Pulsed ICR uses a single-frequency rf excitation, whereas a scanned frequency is used in
FT-ms. A voltage 1-5 V is used to trap +ions. The grid is used for pulsing the ion beam.
B to front and back plates of cell
4:17 AM 5/4/2013 81
Generally equipped with a trapped ion analyzer cell.
Gaseous sample molecules are ionized in the center of the cell by
electrons that are accelerated from the filament through the cell to a
collector plate.
A pulsed voltage applied at the grid serves as a gate to switch the
electron beam on and off periodically.
The ions are held in the cell by a 1 to 5 V potential applied to the trap
plate.
The ions are accelerated by a radio-frequency signal applied to a
transmitter plate (exited plate).
The receiver (collector) plate is connected to a preamplifier to amplify
the image current.
Ions could be stored for several minutes.
The dimensions of the cell are not critical (usually a few cm) on a side.



4:17 AM 5/4/2013 82
Fourier Transform M.S.
Mass analysis performed by detecting cyclotron frequencies of
ions (depend on m/z) in uniform B, in time domain. Then F.T. to
frequency domain (mass spectrum).

Advantages:
No slits or ion optic lenses to adjust.
All ions detected simultaneously for a single ionizing pulse.
Can produce very high mass resolution using slow scan.
e.g. m/m ~ 220000 for m/z = 84 at B = 1.2T
Detection sensitivity independent of m.
Resolution 1/m ( B).

4:17 AM 5/4/2013 83
FT-ms sequence of operations
1. Sample (plus R for CI) admitted to chamber.
2. Short e pulse causes EI/CI of sample.
3. Ions trapped by strong magnetic field, inducing ion
cyclotron motion.
4. Then RF sweep (1 ms) coherently excites all m/z to larger
r.
5. Then decay of cyclotron motion induces image currents in
receiver circuit in cell walls. Observed for ms to s.
6. Quench pulse expels ions.
7. Decay signal in 5 amplified, phase-detected, filtered,
digitized, stored. FT from time to frequency domain to get
mass spectrum.
4:17 AM 5/4/2013 84
Frequency = w/(2) = Bz/(2m) s
-1
Cyclotron frequency depends on m/z for fixed B.
apply V (rf) for t s
Radius expands
under V(rf) to
r = V(rf)t/(2dB)
Principle of ICR spectrometer. Ion A has proper m/z, with v same as rf. It
absorbs energy and describes an orbit of increasing r while keeping the
same v. Ion B has different m/z and does not absorb energy. Ions of same
m/z have same v.
4:17 AM 5/4/2013 85
Ion image current
generation
High vac. So
not FAB
few ms; v varied
linearly
what is B/z range?
FT-ICR
Rapid scan ICR
Ions subjected to slower scan. When v
matches cyclotron resonance frequency
of ion it is excited and detected.
wide-range
ms
4:17 AM 5/4/2013 86
Applications of Molecular Mass Spectrometry: The applications are
numerous and widespread

4:17 AM 5/4/2013 87
Identification of pure compounds
Several kinds of data obtained from the spectrum of a pure compound.
1- MWt of the compound.
2- Molecular formula of the compound.
3- presence or absence of various functional groups (from fragmentation).
Molecular weights from mass spectra:
By identification of the ion peak or (M+1)
+
or the (M-1)
+
peak.
Caution: when electron-impact source is used the molecular ion peak may be
absent or small relative to impurity peaks.
Molecular formula from exact molecular weights:
The molecular ion peak can provide the exact mass.
Requires high resolution instrument which capable of detecting mass
differences of a few thousands of mass unit.
Example:
Molecular ions m/z of : Purine (m =120.044), benzamidine C
7
H
8
N
2
(m
=120.069), ethyltoluene C
9
H
12
(m = 120.096) and acetophenon C
8
H
8
O
(m=120.058).


4:17 AM 5/4/2013 88
If the measured mass of the molecular ion peak is 120.070 (0.005) the all but
C
7
H
8
N
2
are excluded as possible formula. (precision ~ 40 ppm) which
achieved by high resolution double focusing instruments.
Molecular formulas from isotopic ratios
Data from a low resolution instruments can yield useful information about the
formula of a compound if the molecular ion peak is sufficiently intense that
its height and the heights of the (m+1)
+
and (M+2)
+
isotope can be
determined accurately.
Example:
Calculate the ratios of the (m+1)+ to M
+
peak heights for the following two
compounds: dinitrobenzene, C
6
H
4
N
2
O
4
(m = 168) and olefin C
12
H
24
(m
=168).
13
C isotope = 1.08%
For every 100
12
C there is 1.08
13
C atoms.
In nitrobenzene C carbon present so 6x1.08 = 6.48 molecules having one
13
C
for every 100 molecules having non.
S (m+1)
+
peak will be 6.48% of the M
+
peak.
For other isotopic elements:
4:17 AM 5/4/2013 89
MS Isotopes Abundance
Most Other Percentage (%)
H
1
H
2
0.015
C
12
C
13
1.08
N
14
N
15
0.37
S
32
S
33
0.8
S
34
4.4
Cl
35
Cl
37
32.5
Br
79
Br
81
98.0
Si
28
Si
29
5.1
Si
30
3.4
4:17 AM 5/4/2013 90
13
C 6x1.08 = 6.48%
2
H 4x0.015 = 0.060%
15
N 2x0.37 = 0.74%
17
O 24x0.04 = 0.16%

So (M+1)+/M
+
= 7.44%

For C
12
H
24

13
C 12x1.08 = 12.96%
2
H 24x0.015 =0.36%
So (M+1)
+
/M
+
= 13.32%

Thus measuring the heights of (M+1)
+
and M
+
the two compounds can be
discriminated (same MWt.)
4:17 AM 5/4/2013 91
Determination of Molecular Formulas and Molecular Weights
The Molecular Ion and Isotopic Peaks
The presence of heavier isotopes one or two mass units above the
common isotope yields small peaks at M
+.
+1 and M
+.
+2


The intensity of the M
+.
+1 and M
+.
+2 peaks relative to the M peak
can be used to confirm a molecular formula
Example: In the spectrum of methane one expects an M
+.
+1 peak of
1.17% based on a 1.11% natural abundance of
13
C and a 0.016%
natural abundance of
2
H
4:17 AM 5/4/2013 92
Structural information from fragmentation
It is seldom possible or (desirable) to account for all the peaks in the spectrum.
Instead characteristic patterns of fragmentation are sought.

4:17 AM 5/4/2013 93
High-Resolution Mass Spectrometry
Low-resolution mass spectrometers measure m/z
values to the nearest whole number
High-resolution mass spectrometers measure m/z
values to three or four decimal places
The high accuracy of the molecular weight
calculation allows accurate determination of the
molecular formula of a fragment
Example
One can accurately pick the molecular formula of a
fragment with a nominal molecular weight of 32 using
high-resolution MS
4:17 AM 5/4/2013 94
The exact mass of certain nuclides is shown
below
4:17 AM 5/4/2013 95
Fragmentation
In EI mass spectrometry the molecular ion is highly energetic and
can break apart (fragment)
Fragmentation pathways are predictable and can be used to
determine the structure of a molecule
The processes that cause fragmentation are unimolecular
The relative ion abundance is extremely important in predicting
structures of fragments
Fragmentation by Cleavage at a Single Bond
Cleavage of a radical cation occurs to give a radical and a cation
but only the cation is observable by MS
In general the fragmentation proceeds to give mainly the most
stable carbocation
In the spectrum of propane the peak at 29 is the base peak
(most abundant) 100% and the peak at 15 is 5.6%
4:17 AM 5/4/2013 96
Fragmentation Equations
The M
+.
Ion is formed by loss of one of its most loosely held
electrons
If nonbonding electron pairs or pi electrons are present, an
electron from one of these locations is usually lost by electron
impact to form M
+.
Loosely held nonbonding electrons on nitrogen and oxygen,
and t electrons in double bonds are common locations for an
electron to be lost (i.e., where the remaining unshared electron
in M
+.
resides)

In molecules with only C-C and C-H bonds, the location of the lone
electron cannot be predicted and the formula is written to reflect this
using brackets
4:17 AM 5/4/2013 97
Example: The spectrum of hexane

4:17 AM 5/4/2013 98
Example: spectrum of neopentane
Fragmentation of neopentane shows the propensity of cleavage
to occur at a branch point leading to a relatively stable
carbocation
The formation of the 3
o
carbocation is so favored that almost no
molecular ion is detected
4:17 AM 5/4/2013 99
Fragmentation by Cleavage of 2 Bonds
The products are a new radical cation and a neutral molecule
Alcohols usually show an M
+.
-18 peak from loss of water








Cycloalkenes can undergo a retro-Diels Alder reaction (section
13.11) to yield an alkadienyl radical cation




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Compounds identification from comparison spectra
1- Determination of MWt. and isotopic distribution and fragmentation patterns.
2- Narrowing the possible structures.
3- Comparing the unknown spectrum with the available reference compounds.
Procedure assumptions:
1-mass fragmentation patterns are unique.
2- Controlling experimental conditions to produce reproducible spectra.
The EI ionization is the method of choice for spectral comparison.
Limitations:
Heights of mass spectral peaks dependent upon:
E of electron beam.
Location of the sample with respect to the beam.
Sample pressure and temperature.
General geometry of the mass spectrometer.
Generally it is desirable to confirm the identity of a compound by comparison
of its spectrum to the spectrum of an authentic (standard) compound
obtained with the same instrument under identical conditions.

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Hyphenated Mass Spectral Methods
Mass spectrometers are coupled with various efficient separation devices.
Used to analyze mixtures.
Examples:
GC/MS
LC/MC
Capillary electrophoresis/MS
Tandem mass spectrometry (MS/MS).

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Tandem Mass Spectrometry (MS/MS)
Coupling one mass spectrometer to a second.
The first one serves to isolate the molecular ions of various components of a
mixture.
The second one used to fragment each molecular ion one at a time to give a
series of mass spectra.
The first MS equipped with a soft ionization source (CI) the output is a large
molecular ion or protonated molecular ion.
These ions then pass into an ion source for the second spectrometer.
Further fragmentation of the former occurs to give numerous daughter ions
which scanned by the second MS.
Consider a hypothetical mixture of isomers ABCD and BCDA and other
molecules such as UKL and UMN.
1- Singly charged molecular ions obtained by the first MS.
2- Ions with an m/z value corresponding to ABCD
+
and BCDA+ (with
identical m/z) are transmitted to the second MS.


4:17 AM 5/4/2013 103
So molecular ions of isomers are separated from other components of the
mixture.
In the second MS ionization chamber fragmentation takes place and
different daughter ions produced: AB
+
, CD
+
, BC
+
, DA
+
.
Each fragment has a unique m/z so identification is possible in the second
MS analyzer.
The following using MS/Ms :
The analyte consists of two different compounds that have identical masses
(278).
The first spectrometer set to mass of the protonated parent ions.
The two quite different daughter ion spectra obtained after further ionization
by collision and passage through the second spectrometer.

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Example of Daughter Ion MS/Ms Spectra
4:17 AM 5/4/2013 105
Parent ion MS/Ms
The first spectrometer is scanned while the second spectrometer is set to
the mass of one of the daughter ions.
Used to measure the identity and concentration of members of a class of
closely related compounds .
Example:
Determination of alkylphenols (HOC
6
H
4
CH
2
R) in solvent refined coal.
The second spectrometer is set at m/z value of 107 which corresponds to the
ion HOC
6
H
4
CH
2
+
.
The sample is then scanned with the first spectrometer.
All of the alkylphenols in the samples yield an ion of mass 107 regardless of the
nature of R. (measured in a complex sample)
4:17 AM 5/4/2013 106
Instrumentation for Tandem MS
Made up of various combinations of : magnetic sectors, electrostatic
sectors, and quadrupole filter separators.
Consists of:
First type
Magnetic sector
Then an electrostatic sector
Another type:
Two double focusing mass spectrometers each made up of an electrostatic and
a magnetic sector.
Most widely used tandem mass spectrometer has three quadruple filters.
Sample introduced into CI soft source.
The ions are then accelerated into first stage parent ion separator Q1 filter.
The separated ions rapidly moved to Q2 which is a collision chamber so further
ionization of parent ions occurs, dc potential is applied across the rods.
The resulting daughter ions pass Q3 where they scanned and recorded in the
usual way.

4:17 AM 5/4/2013 107
Schematic of Tandem Quadrupole MS/MS
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5
th
edition, Saunders College Publishing.
4:17 AM 5/4/2013 108
Applications of Tandem
Analysis of complex mixtures either organic or biological.
Faster than GC/MS separation complete in milliseconds.
No dilution with mobile phase is needed (interferences occurs) as that of
GC/MS and LC/MS.
So tandem MS is more sensitive than GC/MS or LC/MS (smaller noises).
Used for quant. And qualit. determination of the components of a wide
variety of complex materials either in nature or industry.
Examples:
identification and determination of drug metabolites, insect pheromones,
alkaloids in plants, trace contaminants in air , alkaloids in plants, polymer
sequences, petrochemicals, polychlorinated biphenyls, prostaglandins,
diesel exhausts and odors in air.
It will find wider applications in future.
Disadvantage:
Greater cost.



4:17 AM 5/4/2013 109
Quantitative Application
Fall into two categories:
1- Quantitative determination of molecular species in organic, biological,
and inorganic samples.
2- Determination of the concentration of elements in inorganic and less
commonly organic and biological samples.
Quantitative Determination of Molecular Species
Performed by passage of the sample through a chromatographic or capillary
electrophoretic column and into the spectrometer.
Spectrometer set at suitable m/z value, the ion current is then recorded as a
function of time (selected ion monitoring technique).
Some techniques monitoring occurs at 3 or 4 m/z values.
The plot of data consists of a series of peaks with each appearing at a time
that is characteristic of one of the several components of the sample.
Generally the Area o to concentration of component. (MS detector).



4:17 AM 5/4/2013 110
Second type: analyte conc. are obtained directly from the heights of the mass
spectral peaks.
For simple mixtures it is sometimes possible to find peaks at unique m/z values
for each component.
Calibration curves of peak heights versus concentration used for unkn.
Analysis.
Internal standard of fixed amount (for both sample and standard) is used to
obtain more accurate results. (reduce prep. uncertainty)
Plot of (peak In. of analyte/ peak In. of standard) versus analyte conc.
Types of internal standards:
1- isotopically label analog .
2- Homolog of the analyte that yield intense peak of fragment similar to analyte
fragment.
Precision: 2-10 % relative.
Accuracy depends upon complexity of the mixture:
For gaseous hydrocarbon mixtures: (5-10 components) absolute error: 0.2-0.8
mol% appear.
4:17 AM 5/4/2013 111
Applications:
Mixture without sample heating:
Natural gas: C
3
-C
4
hydrocarbons, C
6
-C
8
saturated hydrocarbons, C
1
-C
4

chlorides and iodides, fluorocarbons, thiophenes, atmospheric pollutants
exhaust gases,,,,,,.
Employing higher temperatures:
C
16
-C
27
alcohols, aromatic acids and esters, steroids, fluorinated polyphenyls,
aliphatic amides, halogenated aromatic derivatives and aromatic nitriles.
High MWt. polymeric materials:
The sample is first pyrolyzed
The volatile products are then admitted into the spectrometer for examination.
Or heating can be performed on the probe of a direct inlet system.
Polymers yield single fragment:
Isoprene (from natural rubber)
Styrene (from polyester).
Ethylene (from polyethylene).
Polymers yield two products:
Depend on amount and kind of pyrolysis temperature.