HANDOUT
SPECTROSCOPY ANALYSIS OF
CHEMISTRY
bY:
Drs. Tri Santoso ,M.Si
PROGRAM STUDI PENDIDIKAN KIMIA
JURUSAN MATEMATIKA DAN ILMU PENGETAHUAN ALAM
FAKULTAS KEGURUAN DAN ILMU PENDIDIKAN
MARCH, 2010
1
I.
Introduction:
Modern spectroscopic methods have largely replaced chemical tests as the standard means of
identifying chemical structures, and for a practising practical organic chemist 1H-NMR has
become a routine tool for identifying the products of reactions.
Spectroscopic techniques are based on the absorption of specific forms of energy by a
molecule and monitoring the affect that this has on the molecule.
Spectroscopy:
Theoretical Background:
Since spectroscopy is based on the interaction of electromagnetic radiation (EMR) with a
molecule, an understanding of electromagnetic radiation is a must.
Spectroscopy monitors the changes in energy states of a molecule, so one must be familiar
with the important energy states and concept of quantisation of energy within a molecule.
Electromagnetic Radiation (EMR):
The part of the electromagnetic radiation spectrum that you are most familar with is "visible
light" but this is just a small portion of all the possible types.
Can you think of common applications of other regions of the electromagnetic spectrum ?
Electromagnetic radiation has both particle and wave properties. Can you think of an
example of each ?
Wave-like properties:
It is important to understand wavelength and frequency and how they relate to one another.
Particle-like properties:
A particle of energy is called a photon. Each photon has a discrete amount of energy : a
quantum.
2
Energy States:
There are many energy 'states' in a molecule. Of particular interest to the organic chemist
will be those relating to the energy associated with the nuclear spin state, the vibration of a
bond or an electronic energy levels (orbitals)
Quantisation of Energy:
The absorption of energy causes an atom or molecule to go
from an initial energy state (the ground state) to another higher
enrgy state (an excited state) The energy changes are
frequently described using an energy level diagram.
The energy states are said to be quantised because there are
only certain values that are possible, there is not a continuous
spread of energy levels available.
Spectroscopic methods:
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II.
Ultra-Violet (UV-VIS)
Infra red (IR)
Nuclear Magnetic Resonance (1H-NMR and 13C-NMR)
Mass Spectrometry (MS)
Getting Structures fromSpectra
Ultraviolet-Visible (uv-vis) Spectroscopy
Basics
Ultraviolet-visible spectropscopy (uv = 200-400 nm, visible = 400-800 nm) corresponds to
electronic excitations between the energy levels that correspond to the molecular orbitals of
the systems. In particular, transitions involving  orbitals and lone pairs (n = non-bonding)
are important and so uv-vis spectroscopy is of most use for identifying conjugated systems
which tend to have stronger absorptions.
The lowest energy transition is that between the highest occupied molecular orbital (HOMO)
and the lowest unoccupied molecular orbital (LUMO) in the ground state. The absorption of
the EM radiation excites an electron to the LUMO and creates an excited state. The more
highly conjugated the system, the smaller the HOMO-LUMO gap, E, and therefore the
lower the frequency and longer the wavelength, . The colours we see in inks, dyes, flowers
etc. are typically due to highly conjugated organic molecules. The unit of the molecule that
is responsible for the absorption is called the chromophore, of which the most common are
C=C ( to *) and C=O (n to *) systems.
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The following table contains some data for polyenes and demonstrates how the wavelength of
the absorbance increases as the conjugated system becomes more extended.
H(CH=CH)nH max / nm
1
170
2
217
3
258
max
15,000
21,000
35,000
Terminology
Absorbance
A, a measure of the amount of radiation that is absorbed
Band
Term to describe a uv-vis absorption which are typically broad.
Chromophore
Structural unit responsible for the absorption.
Molar absorptivity
, absorbance of a sample of molar concentration in 1 cm cell.
Extinction coefficicent An alternative term for the molar absorptivity.
Path length
l, the length of the sample cell in cm.
Beer-Lambert Law
A = c.l
max
The wavelength at maximum absorbance
max
The molar absorbance at max
HOMO
Highest Occupied Molecular Orbital
LUMO
Lowest Unoccupied Molecular Orbital
(c = concentration in moles / litre)
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III. Infra Red Spectrophometry
Basics:
Infra red (IR) spectroscopy deals with the interaction between a molecule and radiation from
the IR region of the EM spectrum (IR region = 4000 - 400 cm-1). The cm-1 unit is the wave
number scale and is given by 1 / (wavelength in cm).
IR radiation causes the excitation of the vibrations of covalent bonds within that molecule.
These vibrations include the stretching and bending modes.
An IR spectrum show the energy absorptions as one 'scans' the IR region of the EM
spectrum. As an example, the IR spectrum of butanal is shown below.
In general terms it is convienient to split an IR spectrum into two approximate regions:


4000-1000 cm-1 known as the functional group region, and
< 1000 cm-1 known as the fingerprint region

Most of the information that is used to interpret an IR spectrum is obtained from the
functional group region.
In practice, it is the polar covalent bonds than are IR "active" and whose excitation
can be observed in an IR spectrum.
In organic molecules these polar covalent bonds represent the functional groups.
Hence, the most useful information obtained from an IR spectrum is what
functional groups are present within the molecule (NMR spectroscopy typically
gives the hydrocarbon fragments).
Remember that some functional groups can be "viewed" as combinations of different
bond types. For example, an ester, CO2R contains both C=O and C-O bonds, and
both are typically seen in an IR spectrum of an ester.
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In the fingerprint region, the spectra tend to be more complex and much harder to
assign.
MOST IMPORTANT THING TO REMEMBER.....
When analysing an IR spectrum avoid the temptation to try to assign every peak.
The fingerprint region, however, can be useful for helping to confirm a structure by direct
comparison with a known spectra.
Infra-Red (IR) Spectroscopy
Hookes' Law
To help understand IR, it is useful to
compare a vibrating bond to the physical
model of a vibrating spring system. The
spring system can be described by
Hooke's Law, as shown in the equation
given on the left.
Consider a bond and the connected
atoms to be a spring with two masses
attached.
Using the force constant k (which
reflects the stiffness of the spring) and
the two masses m1 and m2, then the
equation indicates how the frequency, ,
of the absorption should change as the
properties of the system change.
Consider the following trends:
1. for a stronger bond (larger k value),  increases.
As examples of this, in order of increasing bond strength compare:
CC bonds: C-C (1000 cm-1), C=C (1600 cm-1) and CC (2200 cm-1),
CH bonds: C-C-H (2900 cm-1), C=C-H (3100 cm-1) and CC-H (3300 cm-1),
(n.b. make sure that you understand the bond strengths order)
2. for heavier atoms attached (larger  value),  decreases.
As examples of this, in order of increasing reduced mass compare:
C-H (3000 cm-1)
C-C (1000 cm-1)
C-Cl (800 cm-1)
C-Br (550 cm-1)
C-I (about 500 cm-1)
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The following diagram reflects some of the trends that can be accounted for using Hookes'
Law. It also gives an approximate outline of where specific types of bond stretches may be
found.
Infra-Red (IR) Spectroscopy
Important absorptions:
The more important absorptions that you should probably learn to recognise, in order of
importance are:
Base
Value
Strength /
Shape
Comments
1 C=O
2 O-H
1715
s, "finger"
Exact position depends on type of carbonyl
3600
s, brd
Broad due to H bonding
3 N-H
4 C-O
3500
m
Can tell primary (two peaks) from secondary (one peak)
1100
s
Also check for OH and C=O
5 C=C
1650
w alkene
m-s aromatic
Alkene weak due to low polarity
Aromatic usually in pairs
6 CC
2150
w, sharp
Most obvious in terminal alkynes
7 C-H
3000
s
As hybridisation of C changes sp3-sp2-sp, the
frequency increases
8 CN
2250
m, sharp
Characteristic since little else around it
Bond
abbreviations : "s" = strong, "m" = medium and "w" = weak
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If you know these, then you can identify most of the functional groups of interest. Note that it
is rarely useful to look for C-C since the large majority organic molecules will have them.
You should also be aware that the exact substitution pattern of a particular bond causes shifts
in the position of the absorption and, therefore, ranges of values are typically given in most
tables.
It is possible to rationalise the shifts of absorbances based on electronic effects due to
proximal groups, conjugation and / or ring strain.
In general, when you are trying to work out what a molecule is, you will not just have the IR
spectrum, but you will have other information as well, such as the formula or most likely the
NMR. Always cross check between these sets of information. For example, if the molecular
formula indicates only one O, then you can not have an ester (CO2R) or a carboxylic acid
(CO2H) !
Sample IR Spectra :
By looking at IR spectra that contain known functional groups and comparing and
contrasting them with other IR spectra, one can develop the skills required to be able to
"interpret" an "unknown" IR spectra. Remember that for an organic chemist, the primary role
of IR is to identify the functional groups that are present. A few examples reflecting some of
the more important functional groups are provided below.
Compare them to try to appreciate the subtle differences, comparing frequency, intensity and
shape.
In the first example, of the aromatic hydrocarbon, toluene, we can see both the aromatic and
aliphatic CH stretches, and two absorptions for the aromatic C=C stretches.
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Acetone (2-propanone) is the "classic" carbonyl containing compound with the obvious C=O
stretch in the middle of the spectra. Note that the peak is a very strong absorption. Compare it
with the C=C in the previous case which are weaker and sharper.
The characteristic absorption of the alcohol, 2-propanol, is the broad band due to the
hydrogen bonded -OH group.
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Carboxylic acids contain both C=O and OH groups. Note the broadness of both absorptions
due to the hydrogen bonding and that the C=O is typically at slightly lower frequency than
that of a ketone.
An ester has the following key absorptions, the C=O and typically two bands for the C-O (not
always easy to identify) since there are both sp3 C-O and sp2 C-O bonds.
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IV. Nuclear Magnetic Resonance (NMR) Spectroscopy
Basics:
Nuclei with an odd mass or odd atomic number have "nuclear spin" (in a similar fashion to
the spin of electrons). This includes 1H and 13C (but not 12C). The spins of nuclei are
sufficiently different that NMR experiments can be sensitive for only one particular isotope
of one particular element. The NMR behaviour of 1H and 13C nuclei has been exploited by
organic chemist since they provide valuable information that can be used to deduce the
structure of organic compounds. These will be the focus of our attention.
Since a nucleus is a charged particle in motion, it will develop a magnetic field. 1H and 13C
have nuclear spins of 1/2 and so they behave in a similar fashion to a simple, tiny bar
magnet. In the absence of a magnetic field, these are randomly oriented but when a field is
applied they line up parallel to the applied field, either spin aligned or spin opposed. The
more highly populated state is the lower energy spin state spin aligned situation. Two
schematic representations of these arrangements are shown below:
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In NMR, EM radiation is used to "flip" the
alignment of nuclear spins from the low
energy spin aligned state to the higher
energy spin opposed state. The energy
required for this transition depends on the
strength of the applied magnetic field (see
below) but in is small and corresponds to
the radio frequency range of the EM
spectrum.
As this diagram shows, the energy required for the spin-flip
depends on the magnetic field strength at the nucleus. With
no applied field, there is no energy difference between the
spin states, but as the field increases so does the separation
of energies of the spin states and therefore so does the
frequency required to cause the spin-flip, referred to as
resonance.
The basic arrangement of an
NMR spectrometer is shown
to the left. The sample is
positioned in the magnetic
field and excited via
pulsations in the radio
frequency input circuit. The
realigned magnetic fields
induce a radio signal in the
output circuit which is used
to generate the output
signal. Fourier analysis of
the complex output
produces the actual
spectrum. The pulse is
repeated as many times as
necessary to allow the
signals to be identified from
the background noise.
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Chemical Shift
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

An NMR spectrum is a plot of the radio frequency applied against absorption.
A signal in the spectrum is referred to as a resonance.
The frequency of a signal is known as its chemical shift.
The chemical shift in absolute terms is defined by the frequency of the resonance
expressed with reference to a standard compound which is defined to be at 0 ppm. The
scale is made more manageable by expressing it in parts per million (ppm) and is
indepedent of the spectrometer frequency.
It is often convienient to describe the relative positions of the resonances in an NMR
spectrum. For example, a peak at a chemical shift, , of 10 ppm is said to be downfield
or deshielded with respect to a peak at 5 ppm, or if you prefer, the peak at 5 ppm is
upfield or shielded with respect to the peak at 10 ppm.
Typically for a field strength of 4.7T the resonance frequency of a proton will occur
around 200MHz and for a carbon, around 50.4MHz. The reference compound is the
same for both, tetramethysilane (Si(CH3)4 often just refered to as TMS).
What would be the chemical shift of a peak that occurs 655.2 Hz downfield of TMS on
a spectrum recorded using a 90 MHz spectrometer ?
At what frequency would the chemical shift of chloroform (CHCl3, =7.28 ppm) occur
relative to TMS on a spectrum recorded on a 300 MHz spectrometer ?
A 1 GHz (1000 MHz) NMR spectrometer is being developed, at what frequency and
chemical shift would chloroform occur ?
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Shielding in H-NMR
The magnetic field experienced by a proton is influenced by various structural factors.
Since the magnetic field strength dictates the energy separation of the spin states and hence
the radio frequency of the resonance, the structural factors mean that different types of
proton will occur at different chemical shifts. This is what makes NMR so useful for
structure determination, otherwise all protons would have the same chemical shift.
The various factors include:



inductive effects by electronegative groups
magnetic anisotropy
hydrogen bonding
Electronegativity
The electrons around the proton create a magnetic field that opposes the applied field. Since
this reduces the field
experienced at the nucleus, the electrons are said to shield the proton. It can be useful to think
of this in terms of vectors....
Since the field experienced by the proton
defines the energy difference between the
two spin states, the frequency and hence the
chemical shift, /ppm, will change
depending on the electron density around the
proton. Electronegative groups attached to
the C-H system decrease the electron density
around the protons, and there is less
shielding (i.e. deshielding) so the chemical
shift increases. This is reflected by the plot
shown in the graph to the left which is based
on the data shown below.
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Compound,
CH3X
CH3F
CH3OH
CH3Cl
CH3Br
CH3I
CH4
(CH3)4Si
X
F
O
Cl
Br
I
H
Si
Electronegativity
of X
4.0
3.5
3.1
2.8
2.5
2.1
1.8
Chemical shift, 
/ ppm
4.26
3.4
3.05
2.68
2.16
0.23
0
CH4
CH3Cl
CH2Cl2
CHCl3
0.23
3.05
5.30
7.27
These effects are cumulative, so the
Compound
presence of more electronegative
groups produce more deshielding
 / ppm
and therefore, larger chemical shifts.
These inductive effects at not just
felt by the immediately adjacent
protons as the disruption of electron
density has an influence further
H signal -CH2-CH2-CH2Br
down the chain. However, the effect
does fade rapidly as you move away  / ppm 1.25 1.69 3.30
from the electronegative group. As
an example, look at the chemical
shifts for part of a primary bromide
Magnetic Anisotropy
The word "anisotropic" means "non-uniform". Magnetic anisotropy means that there is a
"non-uniform magnetic field". Electrons in  systems (e.g. aromatics, alkenes, alkynes,
carbonyls etc.) interact with the applied field which induces a magnetic field that causes the
anisotropy. As a result, the nearby protons will experience 3 fields: the applied field, the
shielding field of the valence electrons and the field due to the  system. Depending on the
position of the proton in this third field, it can be either shielded (smaller ) or deshielded
(larger ), which implies that the energy required for, and the frequency of the absorption will
change.
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Hydrogen Bonding
Protons that are involved in hydrogen bonding (this usually means -OH or -NH) are typically
observed over a large range of chemical shift values. The more hydrogen bonding there is,
the more the proton is deshielded and the higher its chemical shift will be. However, since the
amount of hydrogen bonding is susceptible to factors such as solvation, acidity, concentration
and temperature, it can often be difficult to predict.
HINT : It is often a good idea to leave assigning -OH or -NH resonances until other
assignments have been made.
Experimentally, -OH and -NH protons can be identified by carrying out a simple D2O
(deuterium oxide, also known as heavy water) exchange experiment.
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

Run the regular H-NMR experiment
Add a few drops of D2O
Re-run H-NMR experiment
Compare the two spectra a look for peaks that have "disappeared"
Why would a peak disappear ?
Consider the alcohol case for example: R-OH + D2O <=> R-OD + HOD
During the hydrogen bonding, the alcohol and heavy water can "exchange" -H and -D each
other, so the alcohol becomes R-OD.
Although D is NMR active, it's signals are of different energy and are not seen in the H16
NMR, hence the peak due to the -OH disappears. (Note that the HOD will appear...)
Suggest a reason why the acidic protons in a carboxylic acid appear so far downfield (about
12 ppm) ?
H-NMR Chemical shifts
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
The chemical shift is the position on the  scale (in ppm) where the peak occurs.
Typical  /ppm values for protons in different chemical environments are shown in
the figure below.
There are two major factors that influence chemical shifts
(a) deshielding due to reduced electron density (due electronegative atoms) and
(b) anisotropy (due to magnetic fields generated by  bonds).
Note that the figure shows the typical chemical shifts for protons being influenced by a single
group.
In cases where a proton is influenced by more than one group, the effects are essentially
cumulative.
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H-NMR Spectra I
The time has arrived to look at a few H-NMR spectra.....
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Coupling in H-NMR
So far the H-NMR spectra that we have looked at have all had different types of protons that
are seen as singlets in the spectra. This is not the normal case.... spectra usually have peaks
that appear as groups of peaks due to coupling with neighbouring protons, for example, see
the spectra of 1,1-dichloroethane shown below.
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Before we look at the coupling, lets review the assignment of the peaks first:


= 5.9 ppm, integration = 1H deshielded : agrees with the -CHCl2 unit
 = 2.1 ppm, integration = 3H, agrees with -CH3 unit.
Now, what about the coupling patterns ?
Coupling arises because the magnetic field of vicinal (adjacent) protons influences the field
that the proton experiences.
To understand the implications of this we should first consider the effect the -CH group has
on the adjacent -CH3.
The methine -CH can
adopt two alignments
with respect to the
applied field. As a
result, the signal for
the adjacent methyl CH3 is split in two
lines, of equal
intensity, a doublet.
Now consider the effect of the -CH3 group has on the adjacent -CH .
The methyl -CH3
protons give rise to 8
possible combinations
with respect to the
applied field.
However, some
combinations are
equivalent and there
are four magnetically
different effects. As a
result, the signal for
the adjacent methine CH is split into four
lines, of intensity ratio
1:3:3:1, a quartet.
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The proximity of "n" equivalent H on neighbouring carbon atoms, causes the signals
to be split into "n+1" lines.
This is known as the multiplicity or splittingorcoupling pattern of each signal.
Equivalent protons (or those with the same chemical shift) do not couple to each
other.
If the neighbours are not all equivalent, more complex patterns arise.
To a first approximation, protons on adjacent sp3 C tend to behave as if they are
equivalent.
20
Now we can do more a complete analysis, including the application of the "n+1" rule to 1,1dichloroethane:

= 5.9 ppm, quartet, integration = 1H, deshielded
grees with the -CHCl2 unit next to a -CH3 unit (n = 3, so n + 1 = 4 lines)

 = 2.1 ppm, doublet, integration = 3H
grees with -CH3 unit, next to a -CH- (n = 1, so n + 1 = 2 lines)
Coupling Constant, J
The coupling constant, J (usually in frequency units, Hz)
is a measure of the interaction between a pair of protons.
In a vicinal system of the general type, Ha-C-C-Hbthen
the coupling of Ha with Hb, Jab,
MUST BE EQUAL to the coupling of Hb with Ha, Jba,
therefore Jab = Jba.
The implications are that the spacing between the lines
in the coupling patterns are the same as can be seen in
the coupling patterns from the H-NMR spectra of 1,1dichloroethane (see left).
Pascal's Triangle
The relative intensitites of the lines
in a coupling pattern is given by a
binomial expansion or more
conviently by Pascal's triangle.
To derive Pascal's triangle, start at
the apex, and generate each lower
row by creating each number by
adding the two numbers above and
to either side in the row above
together. The first six rows are
shown to the right.
So for H-NMR a proton with zero
neighbours, n = 0, appears as a
single line, a proton with one
neighbours, n =1 as two lines of
equal intensity, a proton with two
neighbours, n = 2, as three lines of
intensities 1 : 2 : 1, etc.
What would the multiplicity and the relative intensitites be for the secondary H in propane ?
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Summary


Individual resonances are split due to coupling with "n" adjacent protons
Number of lines in coupling pattern, L = n + 1
Complex Coupling Patterns
In reality, coupling patterns are often more complex than the simple n+1 rule since the
neighbouring protons are often not equivlalent to each other (i.e. there are different types of
neighbours) and therefore couple differently. In these cases, the "n+1" rule has to be refined
so that each type of neighbour causes n+1 lines.
For example for a proton with two types of neighbour, number of lines, L = (n1 + 1)(n2 + 1).
However, in many cases the lines overlap with each other and the result is further distortion
from the "ideal" pattern.
Coupling patterns invloving aromatic or alkene protons are often complex.
H-NMR Spectra II
Now for a few more spectra, this time where there are coupling patterns.....
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23
Interpretting 1H-NMR Spectra
Let's summarise what can be obtained from a 1H NMR spectrum:
How many types of H ?
Indicated by how many groups of signals there are
in the spectra
What types of H ?
Indicated by the chemical shift of each group
How many H of each type are there?
Indicated by the integration (relative area) of the
signal for each group.
What is the connectivity ?
Look at the coupling patterns. This tells you what is
next to each group
Chemical shift



The chemical shift is the position on the  scale (in ppm) where the peak occurs.
Typical  /ppm values for protons in different chemical environments are shown in
the figure below.
There are two major factors that influence chemical shifts (a) deshielding due to
reduced electron density (due electronegative atoms) and (b) anisotropy (due to
magnetic fields generated by  bonds).
Note that the figure shows the typical chemical shifts for protons being influenced by a single
group. In cases where a proton is influenced by more than one group, the effects are
essentially cumulative.
Integration


The area of a peak is proportional to the number of H that the peak represents
The integral measures the area of the peak
24

The integral gives the relative ratio of the number of H for each peak
Coupling


The proximity of other "n" H atoms on neighbouring carbon atoms, causes the signals
to be split into "n+1" lines.
This is also known as the multiplicity or splitting of each signal.
Be aware that the exact substitution pattern around a particular H causes changes in the
chemical shift and therefore ranges of values are given in the tables and the above figure.
Having a good "feel" for the typical chemical shifts will save yourself lots of time in
examinations, and avoid confusion.
An example of an H NMR is shown below.
Based on the outline given above the four sets of information we get are:
5 basic types of H present in the ratio of 5 : 2 : 2 : 2 : 3.
These are seen as a 5H "singlet" (ArH), two 2H triplets, a 2H quartet and a 3H triplet. Each
triplet tells us that there are 2H in the adjacent position, and a quartet tells us that there are 3H
adjacent.
(Think of it as the lines you see, L = n + 1, where n = number of equivalent adjacent H)
This tells us we that the peaks at 4.4 and 2.8 ppm must be connected as a CH2CH2 unit.
The peaks at 2.1 and 0.9 ppm as a CH2CH3 unit. Using the chemical shift charts, the H can be
assigned to the peaks as below:
7.2ppm (5H) = ArH ;
4.4ppm (2H) = CH2O;
2.8ppm (2H) = Ar-CH2;
2.1ppm (2H) = O=CCH2CH3 and
0.9ppm (3H) = CH2CH3
25
C-NMR Spectroscopy
It is useful to compare and contrast H-NMR and C-NMR as there are certain differences and
similarities:

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13
C has only about 1.1% natural abundance (of carbon atoms)
C does not exhibit NMR behaviour
13
C nucleus is about 400 times less sensitive than H nucleus to the NMR phenomena
Due to low abundance, we do not usually see 13C-13C coupling
Chemical shift range is normally 0 to 220 ppm
Similar factors affect the chemical shifts in 13C as seen for H NMR
Long relaxation times (excited state to ground state) mean no integrations
"Normal" 13C spectra are "broadband, proton decoupled" so the peaks show as single
lines
Number of peaks indicates the number of types of C
12
Here is the simple correlation table of 13C chemical shifts:
The following information is to be gained from a typical broadband decoupled 13C NMR
spectrum:
Interpretting C-NMR Spectra
The following information is to be gained from a typical broadband decoupled 13C NMR
spectrum:
How many types of C ? Indicated by how many signals there are in the spectra
What types of C ? Indicated by the chemical shift of each signal
26
Here are some examples of 13C-NMR spectra.
27
C-NMR Spectroscopy
It is useful to compare and contrast H-NMR and C-NMR as there are certain differences and
similarities:










13
C has only about 1.1% natural abundance (of carbon atoms)
C does not exhibit NMR behaviour (nuclear spin, I = 0)
As a result, C is about 400 times less sensitive than H nucleus to the NMR phenomena
Due to low abundance, we do not usually see 13C-13C coupling
Chemical shift range is normally 0 to 220 ppm
Chemical shifts measured with respect to tetramethylsilane, (CH3)4Si (i.e. TMS)
Similar factors affect the chemical shifts in 13C as seen for H NMR
Long relaxation times (excited state to ground state) mean no integrations
"Normal" 13C spectra are "broadband, proton decoupled" so the peaks show as single
lines
Number of peaks indicates the number of types of C
12
The general implications of these points are that 13C take longer to acquire, though they tend
to look simpler. Overlap of peaks is much less common than for H-NMR which makes it
easier to determine how many types of C are present.
Here is the simple correlation table of 13C chemical shifts:
C=O indicates aldehydes and ketones
O=C-X indicates carbpxylic acids and derivatives such as esters and amides
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V.
Mass Spectroscopy (MS)
Basics
Mass spectrometry is based on slightly different principles to the other spectroscopic
methods.
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 a radical 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.

Probably the most useful information you should be able to obtain from a MS
spectrum is the molecular weight of the sample.
29

This will often be the heaviest ion observed from the sample provided this ion is
stable enough to be observed.
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 cleave of bonds at successive -CH2- units
The MS of benzyl alcohol is shown below. The molecular ion is seen at m/z = 108.
Fragmentation via loss of 17 (-OH) gives a common fragment seen for alkyl benzenes at m/z
= 91. Loss of 31 (-CH2OH) from the molecular ion gives 77 corresponding to the phenyl
cation. Note the small peaks at 109 and 110 which correspond to the presence of small
amounts of 13C in the sample (which has about 1% natural abundance).
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Isotope patterns





Mass spectrometers are capable of separating and detecting individual ions even those
that only differ by a single atomic mass unit.
As a result molecules containing different isotopes can be distinguished.
This is most apparent when atoms such as bromine or chlorine are present (79Br : 81Br,
intensity 1:1 and 35Cl : 37Cl, intensity 3:1) where peaks at "M" and "M+2" are obtained.
The intensity ratios in the isotope patterns are due to the natural abundance of the
isotopes.
"M+1" peaks are seen due the the presence of 13C in the sample.
The following two mass spectra show examples of haloalkanes with characteristic isotope
patterns.
The first MS is of 2-chloropropane. Note the isotope pattern at 78 and 80 that represent the M
amd M+2 in a 3:1 ratio.
Loss of 35Cl from 78 or 37Cl from 80 gives the base peak a m/z = 43, corresponding to the
secondary propyl cation. Note that the peaks at m/z = 63 and 65 still contain Cl and therefore
also show the 3:1 isotope pattern.
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The second MS is of 1-bromopropane. Note the isotope pattern at 122 and 124 that represent
the M amd M+2 in a 1:1 ratio. Loss of 79Br from 122 or 81Br from 124 gives the base peak a
m/z = 43, corresponding to the propyl cation. Note that other peaks, such as those at m/z =
107 and 109 still contain Br and therefore also show the 1:1 isotope pattern.
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Getting Structures from Spectra
Good problem solving skills are often based on a good method of attack. Here is some
general advice of how to go about determining structures from spectral data, be it from real
spectra of an experimental sample, or in the context of a question. The order of events here
works towards using the H-NMR at the last stage since it is potentially the most useful for
"assembling" the structure.
However, at each stage it is a good idea to use information in other spectra (if available) to
seek support your interpretation or address any doubts. Try to get a molecular formula at the
earliest possible opportunity and then calculate the index of hydrogen deficiency (IHD).
 MS
o determine the molecular weight
o identify the presence of isotopes patterns for Cl or Br
 UV
o is the system conjugated ?
 IR
o identify the functional groups that are present
 13C-NMR
o how many types of carbon ?
o what types of carbon ?
 H-NMR
o
o
o
o
how many types of hydrogen ?
how many of each type ?
what types of hydrogen ?
how are they connected ?
Having completed an analysis of the available spectra,



list the pieces that have been identifed
check the pieces the MW and / or molecular formula and refine the pieces to fit
assemble the pieces paying particular attention to H-NMR chemical shifts and
coupling patterns
Once you think you have the answer, check it with the H-NMR very carefully... it is probably
the most critical test to pass.
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VI. Atomic Absorption Spectroscopy
The study of absorption spectra by means of passing electromagnetic radiation through an
atomic medium that is selectively absorbing; this produces pure electronic transitions free
from vibrational and rotational transitions
(Academic Press Dictionary of Science and Technology)
1. Introduction.
Figure1. Elements detectable by atomic absorption are highlighted in pink in this periodic
table
Atomic absorption methods measure the amount of energy (in the form of photons of light,
and thus a change in the wavelength) absorbed by the sample. Specifically, a detector
measures the wavelengths of light transmitted by the sample (the "after" wavelengths), and
compares them to the wavelengths, which originally passed through the sample (the "before"
wavelengths). A signal processor then integrates the changes in wavelength, which appear in
the readout as peaks of energy absorption at discrete wavelengths (see schematic of an
atomic-absorption experiment).
Any atom has its own distinct pattern of wavelengths at which it will absorb energy, due to
the unique configuration of electrons in its outer shell. This allows for the qualitative analysis
of a pure sample.
In order to tell how much of a known element is present in a sample, one must first establish
a basis for comparison using known quantities. It can be done producing a calibration curve.
For this process, a known wavelength is selected, and the detector will measure only the
energy emitted at that wavelength. However, as the concentration of the target atom in the
sample increases, absorption will also increase proportionally. Thus, one runs a series of
known concentrations of some compound, and records the corresponding degree of
absorbance, which is an inverse percentage of light transmitted. A straight line can then be
drawn between all of the known points. From this line, one can then extrapolate the
concentration of the substance under investigation from its absorbance. The use of special
light sources and specific wavelength selection allows the quantitative determination of
individual components of a multielement mixture.
The phenomenon of atomic absorption (AA) was first observed in 1802 with the discovery of
the Fraunhofer lines in the sun's spectrum. It was not until 1953 that Australian physicist Sir
34
Alan Walsh demonstrated that atomic absorption could be used as a quantitative analtical
tool. Atomic absorption analysis involves measuring the absorption of light by vaporized
ground state atoms and relating the absorption to concentration. The incident light beam is
attenuated by atomic vapor absorption according to Beer's law.
The process of atomic absorption spectroscopy (AAS) involves two steps:
1. Atomization of the sample
2. The absorption of radiation from a light source by the free atoms
The sample, either a liquid or a solid, is atomized in either a flame or a graphite furnace.
Upon the absorption of ultraviolet or visible light, the free atoms undergo electronic
transitions from the ground state to excited electronic states.
To obtain the best results in AA, the instrumental and chemical parameters of the system
must be geared toward the production of neutral ground state atoms of the element of interest.
A common method is to introduce a liquid sample into a flame. Upon introduction, the
sample solution is dispersed into a fine spray, the spray is then desolvated into salt particles
in the flame and the particles are subsequently vaporized into neutral atoms, ionic species and
molecular species. All of these conversion processes occur in geometrically definable regions
in the flame. It is therefore important to set the instrument parameters such that the light from
the source (typically a hollow-cathode lamp) is directed through the region of the flame that
contains the maximum number of neutral atoms. The light produced by the hollow-cathode
lamp is emitted from excited atoms of the same element which is to be determined. Therefore
the radiant energy corresponds directly to the wavelength which is absorbable by the
atomized sample. This method provides both sensitivity and selectivity since other elements
in the sample will not generally absorb the chosen wavelength and thus, will not interfere
with the measurement. To reduce background interference, the wavelength of interest is
isolated by a monochromator placed between the sample and the detector.
35
2. Instrumentation
Figure 2. Perkin-Elmer Spectrophotometer Model 460
In atomic absorption (see schematic of an atomic-absorption experiment), there are two
methods of adding thermal energy to a sample. A graphite furnace AAS uses a graphite tube
with a strong electric current to heat the sample. In flame AAS (see photo above), we aspirate
a sample into a flame using a nebulizer. The flame is lined up in a beam of light of the
appropriate wavelength. The flame (thermal energy) causes the atom to undergo a transition
from the ground state to the first excited state. When the atoms make their transition, they
absorb some of the light from the beam. The more concentrated the solution, the more
light energy is absorbed!
The light beam is generated by lamp that is specific for a target metal. The lamp must be
perfectly aligned so the beam crosses the hottest part of the flame. The light passed through
the flame is received by the monochromator, which is set to accept and transmit radiation at
the specified wavelength and travels into the detector. The detector measures the intensity of
the beam of light. When some of the light is absorbed by metal, the beam's intensity is
reduced. The detector records that reduction as absorption. That absorption is shown on
output device by the data system.
We can find the concentrations of metals in a sample running a series of calibration standards
through the instrument. The instrument will record the absorption generated by a given
concentration. By plotting the absorption versus the concentrations of the standards, a
calibration curve can be plotted. We can then look at the absorption for a sample solution and
use the calibration curves to determine the concentration in that
3. Techniques of Measurement and EPA Methods Using
FAAS
Atomic absorption spectrometry is a fairly universal analytical method for determination of
metallic elements when present in both trace and major concentrations. The EPA employs
this technique for determining the metal concentration in samples from a variety of matrices.
36
A) Sample preparation
Depending on the information required, total recoverable metals, dissolved metals, suspended
metals, and total metals could be obtained from a certain environmental matrix. Table 1 lists
the EPA method number for sample processing in terms of the environmental matrices and
information required. For more detail information, readers could refer to EPA document SW846 "Test methods for evaluating solid wastes".
Table 1 EPA sample processing method for
metallic element analysis
Analysis Target
total recoverable
metals
dissolved metals
suspended metals
Method
Number
Environmental Matrice
3005
ground water/surface water
3005
3005
ground water/surface water
ground water/surface water
aqueous samples, wastes that contain suspended solids and
mobility-procedure extracts
aqueous samples, wastes that contain suspended solids and
mobility-procedure extracts
aqueous samples, wastes that contain suspended solids and
mobility-procedure extracts
sediments, sludges and soil samples
sludges, sediment, soil and oil
total metals
3010
total metals
3015
total metals
3020
total metals
total metals
3050
3051
Appropriate acid digestion is employed in these methods. Hydrochloric acid digestion is not
suitable for samples, which will be analyzed by graphite furnace atomic absorption
spectroscopy because it can cause interferences during furnace atomization.
B) Calibration and standard curves
As with other analytical techniques, atomic absorption spectrometry requires careful
calibration. EPA QA/QC demands calibration through several steps including interference
check sample, calibration verification, calibration standards, bland control, and linear
dynamic range.
The idealized calibration or standard curve is stated by Beer's law that the absorbance of an
absorbing analyte is proportional to its concentration.
Unfortunately, deviations from linearity usually occur, especially as the concentration of
metallic analytes increases due to various reasons, such as unabsorbed radiation, stray light,
or disproportionate decomposition of molecules at high concentrations. Figure 3 shows an
idealized and deviation of response curve. The curvature could be minimized, although it is
impossible to be avoided completely. It is desirable to work in the linearity response range.
The rule of thumb is that a minimum of five standards and a blank should be prepared in
order to have sufficient information to fit the standard curve appropriately. Manufacturers
should be consulted if a manual curvature correction function is available for a specific
instrument.
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Figure 3. Idealized/deviation response curve
If the sample concentration is too high to permit accurate analysis in linearity response range,
there are three alternatives that may help bring the absorbance into the optimum working
range:
1) sample dilution
2) using an alternative wavelength having a lower absorptivity
3) reducing the path length by rotating the burner hand.
C) EPA method for metal analysis
Flame atomic absorption methods are referred to as direct aspiration determinations. They are
normally completed as single element analyses and are relatively free of interelement spectral
interferences. For some elements, the temperature or type of flame used is critical. If flame
and analytical conditions are not properly used, chemical and ionization interferences can
occur.
Graphite furnace atomic absorption spectrometry replaces the flame with an electrically
heated graphite furnace. The major advantage of this technique is that the detection limit can
be extremely low. It is applicable for relatively clean samples, however, interferences could
be a real problem. It is important for the analyst to establish a set of analytical protocol which
is appropriate for the sample to be analyzed and for the information required. Table 2 lists the
available method for different metal analysis provided in EPA manual SW-846.
Table 2. EPA methods for determination of metals by direct
aspiration
Analyte
aluminum
beryllium
chromium
iron
magnesium
nickel
silver
thallium
zinc
Method
number
7020
7090
7190
7380
7450
7520
7760A
7840
7951
Method
number
7040
7130
7200
7420
7460
7550
7770
7870
Analyte
antimony
cadmium
cobalt
lead
manganese
osmium
sodium
tin
38
Analyte
barium
calcium
copper
lithium
molybdenum
potassium
strontium
vanadium
Method
number
7080A
7140
7210
7430
7480
7610
7780
7910
D) Interferences
Since the concentration of the analyte element is considered to be proportional to the ground
state atom population in the flame, any factor that affects the ground state population of the
analyte element can be classified as interference. Factors that may affect the ability of the
instrument to read this parameter can also be classified as interference. The following are the
most common interferences:
a) Spectral interferences are due to radiation overlapping that of the light source. The
interference radiation may be an emission line of another element or compound, or general
background radiation from the flame, solvent, or analytical sample. This usually occurs when
using organic solvents, but can also happen when determining sodium with magnesium
present, iron with copper or iron with nickel.
b) Formation of compounds that do not dissociate in the flame. The most common example is
the formation of calcium and strontium phosphates.
c) Ionization of the analyte reduces the signal. This is commonly happens to barium, calcium,
strontium, sodium and potassium.
d) Matrix interferences due to differences between surface tension and viscosity of test
solutions and standards.
e) Broadening of a spectral line, which can occur due to a number of factors. The most
common line width broadening effects are:
1. Doppler effect
This effect arises because atoms will have different components of velocity along the
line of observation.
2. Lorentz effect
This effect occurs as a result of the concentration of foreign atoms present in the
environment of the emitting or absorbing atoms. The magnitude of the broadening
varies with the pressure of the foreign gases and their physical properties.
3. Quenching effect
In a low-pressure spectral source, quenching collision can occur in flames as the result
of the presence of foreign gas molecules with vibration levels very close to the excited
state of the resonance line.
4. Self absorption or self-reversal effect
The atoms of the same kind as that emitting radiation will absorb maximum radiation
at the center of the line than at the wings, resulting in the change of shape of the line
as well as its intensity. This effect becomes serious if the vapor, which is absorbing
radiation is considerably cooler than that which is emitting radiation.
39
Atomic Absorption Resources.
EPA document SW-846 "Test methods for evaluating solid wastes".
Textbooks:
Haswell, S.J., 1991. Atomic Absorption Spectrometry; Theory, Design and
Applications. Elsevier, Amsterdam.
Reynolds, R.J. et al., 1970. Atomic Absorption Spectroscopy. Barnes & Noble Inc.,
New York.
Schrenk, W.G., 1975. Analytical Atomic Spectroscopy. Plenum Press, New York.
Varma, A., 1985. Handbook of Atomic Absorption Analysis. Vol. I. CRC Press, Boca
Raton.
Scientific journals related to Atomic Absorption Spectroscopy:
Journal of Analytical Atomic Spectrometry
Published by: Royal Society of Chemistry
Spectrochimica Acta Part B: Atomic Spectroscopy
Published by: Elsevier Science
40