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CH437 ORGANIC STRUCTURE ANALYSIS
CLASS 1: INTRODUCTION AND MASS SPECTROMETRY 1
Synopsis. Introduction to spectroscopic methods used in the determination of organic structures.
Principles of mass spectrometry. Electron impact (EI) and chemical ionization (CI).
Introduction
Structure determination is important throughout chemistry and closely related
subjects. When a new compound has been synthesized or discovered, it is
crucial to have a reliable check on its identity:
Is its structure the same as that of the target molecule?
Is it the same structure as a known natural product or previously
synthesized substance?
What is the structure of this new compound that has been recently isolated
from pine needles, or grape skins or marine sponges and which exhibits
promising therapeutic properties?
These are just some kinds of questions that the application of spectroscopic (and
other) techniques can be used to answer.
Nowadays, spectroscopic methods are overwhelmingly the most important
structure determination techniques, although making derivatives and other
chemical and physical methods can be useful in certain circumstances. These
used to be the core of structure determination, before the development of
spectroscopic methods.
The major spectroscopic techniques in organic chemistry are summarized in the
table below.
Name of method
Type of transition Comments
or process/energy
of
radiation
(electromagnetic
spectrum)
Mass
spectrometry
Ionization
(fragmentation)
Information
obtained
from
spectrum
Includes GC-MS Molar
and HPLC-MS. molecular
mass,
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(MS)
Nuclear
magnetic
resonance
spectroscopy
(NMR)
Electron
resonance
(ESR)*
The former needs
volatile
substances
Nuclear spin (in 1D
and
2D.
presence
of Multinuclear, but
1H and 13C most
magnetic
field)/radiofrequency important
region
spin Electron
spin/microwave
region
Infrared
(IR)
spectroscopy
and
Raman
spectroscopy*
Ultravioletvisible (UV-VIS)
spectrocopy
Vibrational
(rotational)/
region
Electronic/UVvisible region
formulas,
structure
(sometimes)
Structural
skeleton.
Also
dynamic (kinetic)
and
thermodynamic
information
For
radical Structural
(unpaired
skeleton
electron)
determination
Fourier transform Functional groups
IR IR (FTIR) most
common
Including diode
array type for
rapid
data
acquisition
Presence
of
conjugated
or
aromatic systems.
Also quantitative
and
empirical
methods
Circular
Electronic/UVUses
polarized Absolute
dichroism (CD) visible
region, light, usually in configuration
and
optical usually
UV-visible region
rotatory
dispersion (ORD)
X-Ray
Diffraction by atoms Needs
good Molecular
and
crystallography* in crystal lattice
crystals
crystal structure,
hydrogen bonds,
absolute
configuration
*Not considered in this course
Mass Spectrometry 1: Principles and Methods of Ionization
Mass spectrometry is an outstanding technique in today’s analytical methods: it
is a major tool for the determination of organic structures, although it is widely
applied throughout science.
There are many kinds of mass spectrometry, but all rely on the production
of ions, followed by their selection (in the gas phase) by an analyzer and
their subsequent detection. The essential features of a mass spectrometer are
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shown below: the various types of spectrometer vary according to the type of
ionization, the type of analyzer used and the type of detector.
Vacuum or
atmospheric
pressure
Inlet probe
or flow from
GC, HPLC
or CZE
instrument
Vacuum (typically
10-5 or 10-6 torr)
Mass analyzer
where ions are
ions "sorted",
ions
Ion source
according
(ionizer)
to m/z. May be
more than one
analyzer (tandem
MS)
Detector
signal
Computer
Data
(spectrum)
Ionization
The major methods of ionization are:
Electron ionization (EI)
Chemical ionization (CI)
Electrospray ionization (ESI)
Atmospheric pressure chemical ionization (APCI)
Matrix-assisted laser-desorption ionization (MALDI)
ESI and APCI are especially popular with HPLC (HPLC-MS). The above
ionization methods are reviewed in this course, but other methods include:
Fast atom bombardment (FAB), liquid secondary ion mass spectrometry
(LSIMS), thermospray ionization (TSI), field-desorption ionization (FDI), plasma
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desorption ionization (PDI), particle beam ionization (with HPLC), and for
inorganic and organometallic compounds, thermal cavity, spark source, glow
discharge and inductively coupled plasma source ionization.
Ionization can generally produce both positive and negative ions (they are called
ions, but may be, in fact, radical ions – odd electron ions), as illustrated for
electron ionization. Positive ion MS is more common.
electron
ejection
M.+ +
M : + e:M
._
electron
absorption
2e-
positive ion
MS
negative ion
MS
In many cases, especially EI, the production of ions occurs at energies high
enough to cause dissociation (fragmentation) of some of the molecular ions:
N
loss of
neutral
fragment
+
m.+
odd
electron
ion
M.+
molecular
ion (odd
electron)
m+
+
even
electron
ion
R.
loss of
radical
fragment
m.+ and m+ are called fragment or daughter ions
Fragmentation pathways depend on structure and hence much structural
information can be obtained from their study.
Multiply charged ions (all the above examples are of singly charged species) can
also be found in mass spectrometry, especially when ESI and MALDI are the
ionization methods (see later).
Note that although ionization can be carried out at high vacuum (EI, MALDI: 10 -6
torr) or low vacuum (CI: 10-3 torr) or atmospheric pressure (ESI, APCI), ion
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selection and detection must occur at high vacuum. The main reason for this is to
prevent ion-molecule reactions before the ions, produced only by initial ionization
and fragmentation, reach the detector.
Ion Selection
During the course of a mass spectrometric determination, many different ions are
normally produced: ion selection is a kind of sorting of the ions according to
certain physical characteristics, principally their m/z ratios. Selection occurs in
the analyzer part of the instrument: there may be more than one analyzer, as in
tandem MS. The major types of analyzers are:
Dispersive Analyzers (these actually separate ions according to m/z)
Magnetic Sector and Electric Sector (BE)
Quadrupole (Q)
Ion Trap (QIT)
Time-of-Flight (TOF)
Non-Dispersive Analyzers (these sort the ions, without separating them)
Ion Cyclotron Resonance (ICR) (used in Fourier Transform MS, FTMS)
Detectors
Detectors convert the collection of ions into an electric current when ions impact
the detector. Because the number of ions reaching the detector is normally small,
various kinds of amplification systems are used. Detectors fall into two major
categories:
Point ion Collectors – these detect the arrival of ions sequentially at one point.
Array Detectors – these detect arrival of ions simultaneously along a plane.
These two categories may be further subdivided into Faraday Cup detectors and
Electron (Photon) Multiplier detectors. Each type has its advantages and
disadvantages, see “Detectors”.
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Computer and Data System
The computer and data system are very important in modern MS instruments.
The computer not only processes the mass spectral output from the detector, but
also controls each section of the instrument in both real experiments and in
“tuning”. This is summarized below. See also “GC-MS and HPLC-MS” for further
accounts of how the computer operates.
set mass
range (m/z)
Digital input:
keyboard,
mouse
Digital-toanalog
converter
(DAC)
Electron
multiplier
scan 0 - U
volts
RF
generator
Ion abundances
analysis
of masses
to voltages
Analog
scan
voltage
Analog-toComputer
digital
converter
(ADC)
Digitalized calibration
scan voltages
and ion
abundances
Ionization Techniques
This is fundamental to all MS methods: the substance being studied needs to be
ionized and its ions subsequently selected (analyzed) and detected. Because ion
selection and detection is carried out at high vacuum (10 -5 – 10-6 torr), the ions
must be ultimately in the gas phase, but ionization can occur in either condensed
phases or the gas phase. Either the substance must be vaporized prior to its
ionization (as in APCI, EI and CI) or ionized prior to its vaporization (as in ESI) or
vaporized and ionized at the same time (as in MALDI).
Electron Ionization (EI)
This is the oldest ionization technique still in use with commercial mass
spectrometers. Electrons are emitted from a hot filament and are accelerated
through an electric field of potential difference typically 70 V, after which they
have energies of around 70 eV. A typical EI source is shown below.
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Mass
spectrum
%A
m/z
An energetic electron interacts with a sample molecule when it passes close by
or travels through its electron cloud. The usual result is the ejection of an outer
electron from the molecule, producing an odd-electron cation (radical cation),
known simply as the molecular ion:
M:
+
e-

M.+
+
2e-
For a limited number of substances, radical anions are produced by electron
absorption, especially if the electron is less energetic (say, 10 eV) and the
molecule contains groups that stabilize a negative charge:
M
+
e-

M.-
This gives rise to the (less common) negative ion MS: most EIMS is concerned
with positive ions.
Although the energy of the ionizing electrons can be altered, the standard value
is 70 eV – high enough to give some of the molecular ions so much excess
energy that they dissociate (fragment), often by several different routes (called
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fragmentation pathways). The fragment ions produced by dissociation of larger
ions (or molecular ion) are called daughter ions: most fragmentation occurs in the
ion source. A simple scheme is illustrated below.
qM.+ (undissociated molecular ion)
pM.+
rA+ +
sB.+ +
c. (radical)
p = q + r + s,
in abundances
d (molecule)
This gives rise to the following mass spectrum.
E.g. 100%
Ion abundance
or relative ion
abundance
(%)
E.g. 30%
r
E.g. 20%
s
q
m/z
Advantages of EI: Gives much structural information from fragmentation
patterns (EI is called a “hard” form of ionization).
Disadvantages: Sometimes fragmentation is so extensive that M .+ is of very
low abundance or is undetected: this gives lack of formula mass
information.
The sample needs to be volatile and thermally stable, as ionization occurs
at high vacuum.
EI is comparatively inefficient (~0.1% of sample molecules ionized) and so
is less sensitive than others (e.g. ESI)
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Chemical Ionization (CI)
This is essentially high energy EI (typically 200 eV), with a controlled amount of
reagent gas R (typically CH4, NH3 or isobutane) in the ionization chamber, so that
the pressure is typically 10-3 torr, but it can be higher. The typical CI process is
shown below.
R
R.+
+

e-
+
R.+
+
2e-
(1)
(2)
RH

RH+
+
R.
M

MH+
+
R
RH+ +
(3)
The concentration of reagent R is much higher than that of the sample M, so that
electron ionization of reagent molecules is the more likely (1). Reagent molecular
ions undergo reaction with neutral reagent molecules to form RH + (2), which then
protonates sample molecules and regenerates R (3). An example of (2) and (3)
shows CI with CH4, a common reagent gas:
.
CH4 + +
CH4
+
CH5 +
CH5 + +
M
CH4
+
.
CH3
MH+
The last step of CI (protonation of M) is much less energetic than EI and hence
CI is called a “soft ionization” technique. The “molecular ion” (here, MH +)
receives relatively excess energy and so fragmentation is less pronounced than
in EI. Hence CI is especially useful when EI gives a mass spectrum with low or
zero abundance molecular ion, as shown below.
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Electrospray Ionization (ESI)
This mode of ionization is most commonly used with HPLC (including capillary
column HPLC) and CZE, using mixed aqueous organic solvents (e.g.
H2O/MeCN) and (sometimes) added buffer salts. It is an example of atmospheric
pressure ionization (API).
The HPLC or CZE eluent containing the sample is converted to an aerosol at
atmospheric pressure by the action of a high voltage (typically 4 kV for aqueous
solutions) applied to the capillary nebulizer tube (see diagram overleaf). The
strong electric field (~ 106 V/m) not only produces very small droplets but also
induces a high charge accumulation (caused by ionizations) at the droplet
surface.
See K. Hiroaka, and I. Kudaka, Rapid Comm Mass Spectrom, 4, 519 (1990).
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The aerosol is made up many tiny charged droplets, which under the influence of
a strong electric field and a drying flow of warm gas, shrink in stages, losing both
solvent molecules and ions. At some point, a droplet will be too small for the
number of ions it contains: it then disintegrates and discharges its ions (called
ion desorption), the remaining solvent molecules being pumped to vacuum. The
curtain of warm gas helps break up ion clusters. This process is illustrated below
for the positive ion mode. By reversing the polarity of the lens, negative ions,
rather than positive ions may be collected.
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Because ESI is a “soft ionization’ technique, ESI mass spectra posses relatively
little fragmentation, as illustrated below. This is one of its advantages over EI.
Multiple-charged ions are formed if there are many ionizable sites in the
molecule, as in peptides and proteins, so that the formula masses of large
molecules can be determined by ESI – another big advantage over EI. Most
analyzers have limits on the size of m/z that can be measured with acceptable
accuracy. For example, if a sample molecule has M = 10,000, it would be difficult
to measure its m/z value if the ion was merely M.+ or MH+, but if it has 20
ionizable groups it can form [M + 20H]20+ in ESIMS, with a mass of 10, 020/20 =
501; much easier to measure.
Finally, ESI is a more efficient ionization method than EI and hence ESIMS is
more sensitive than EIMS. ESI can be made even more sensitive by the use of a
corona needle in the electrospray: this boosts the production of ions.
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Multiply Charged Ions
One of the original uses of ESI was in mass spectrometric investigation of
proteins, which have many ionizable groups and hence produce multiply charged
ions of the type [M + nH]n+ and [M – nH]n-.
The ESI mass spectra of such compounds usually correspond to the statistical
distribution of consecutive peaks corresponding to the multiply charged ions (e.g.
from [M + 12H]12+ to, say, [M + 20H]20+), as shown for lysozyme, below. There
are usually very few peaks arising from fragmentation or decomposition.
By application of suitable computer algorithms, the molecular mass of the protein
can be determined by (computer) conversion of the multiply charged ion data in
the spectrum to singly charged ion data. An example of how this can be done
manually is discussed next.
For any two ions of the type [M + nH]n+, separated by j – 1 peaks, the charge on
the ion corresponding to the lower m/z value {(m/z)1} can be obtained from
equation 1.
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z1 =
j[(m/z) 2 _ mp]
[(m/z)2
_
(1)
(m/z)1]
mp = mass of H+;
1.0073 Da
The molecular mass (M) of the protein (in Da) is then determined by equation 2:
M = z1[(m/z)1 + mp]
(2)
Several calculations of this kind can be carried out on different pairs of peaks and
thus an average value for the molecular mass can be obtained.
Additionally, high resolution allows direct determination of the charge state of an
ion corresponding to a particular peak in the ESI mass spectrum, because
resolution of the peak shows several peaks (with 1/z observed distance between
them) corresponding to isotope distribution (see class 5).
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