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Fragmentation Patterns in the Mass Spectra of Organic

Compounds
This page looks at how fragmentation patterns are formed when organic molecules
are fed into a mass spectrometer, and how you can get information from the mass
spectrum.

The origin of fragmentation patterns


The formation of molecular ions
When the vaporised organic sample passes into the ionisation
chamber of a mass spectrometer, it is bombarded by a stream of
electrons. These electrons have a high enough energy to knock an
electron off an organic molecule to form a positive ion. This ion is
called the molecular ion - or sometimes the parent ion.
Note: If you aren't sure about how a mass spectrum is
produced, it might be worth taking a quick look at the page
describing how a mass spectrometer works.

The molecular ion is often given the symbol M+ or


- the dot in
this second version represents the fact that somewhere in the ion
there will be a single unpaired electron. That's one half of what was
originally a pair of electrons - the other half is the electron which
was removed in the ionisation process.
Fragmentation
The molecular ions are energetically unstable, and some of them
will break up into smaller pieces. The simplest case is that a
molecular ion breaks into two parts - one of which is another
positive ion, and the other is an uncharged free radical.

Note: A free radical is an atom or group of atoms which contains

a single unpaired electron.


More complicated break-ups are beyond the scope of A'level
syllabuses.

The uncharged free radical won't produce a line on the mass


spectrum. Only charged particles will be accelerated, deflected and
detected by the mass spectrometer. These uncharged particles will
simply get lost in the machine - eventually, they get removed by the
vacuum pump.
The ion, X+, will travel through the mass spectrometer just like any
other positive ion - and will produce a line on the stick diagram.
All sorts of fragmentations of the original molecular ion are possible
- and that means that you will get a whole host of lines in the mass
spectrum. For example, the mass spectrum of pentane looks like
this:

Note: All the mass spectra on this page have been drawn using
data from the Spectral Data Base System for Organic
Compounds (SDBS) at the National Institute of Materials and
Chemical Research in Japan.
They have been simplified by omitting all the minor lines with
peak heights of 2% or less of the base peak (the tallest peak).

It's important to realise that the pattern of lines in the mass


spectrum of an organic compound tells you something quite
different from the pattern of lines in the mass spectrum of an
element. With an element, each line represents a
differentisotope of that element. With a compound, each line
represents a different fragment produced when the molecular ion
breaks up.
Note: If you are interested in the mass spectra of elements, you
could follow this link.

The molecular ion peak and the base peak


In the stick diagram showing the mass spectrum of pentane, the
line produced by the heaviest ion passing through the machine (at
m/z = 72) is due to the molecular ion.
Note: You have to be a bit careful about this, because in some
cases, the molecular ion is so unstable that every single one of
them splits up, and none gets through the machine to register in
the mass spectrum. You are very unlikely to come across such a
case at A'level.

The tallest line in the stick diagram (in this case at m/z = 43) is
called the base peak. This is usually given an arbitrary height of
100, and the height of everything else is measured relative to this.
The base peak is the tallest peak because it represents the
commonest fragment ion to be formed - either because there are
several ways in which it could be produced during fragmentation of
the parent ion, or because it is a particularly stable ion.

Using fragmentation patterns


This section will ignore the information you can get from the
molecular ion (or ions). That is covered in three other pages which

you can get at via the mass spectrometry menu. You will find a link
at the bottom of the page.
Working out which ion produces which line
This is generally the simplest thing you can be asked to do.
The mass spectrum of pentane
Let's have another look at the mass spectrum for pentane:

What causes the line at m/z = 57?


How many carbon atoms are there in this ion? There can't be 5
because 5 x 12 = 60. What about 4? 4 x 12 = 48. That leaves 9 to
make up a total of 57. How about C4H9+ then?
C4H9+ would be [CH3CH2CH2CH2]+, and this would be produced by
the following fragmentation:

The methyl radical produced will simply get lost in the machine.
The line at m/z = 43 can be worked out similarly. If you play around
with the numbers, you will find that this corresponds to a break
producing a 3-carbon ion:

The line at m/z = 29 is typical of an ethyl ion, [CH 3CH2]+:

The other lines in the mass spectrum are more difficult to explain.
For example, lines with m/z values 1 or 2 less than one of the easy
lines are often due to loss of one or more hydrogen atoms during
the fragmentation process. You are very unlikely to have to explain
any but the most obvious cases in an A'level exam.

The mass spectrum of pentan-3-one

This time the base peak (the tallest peak - and so the commonest
fragment ion) is at m/z = 57. But this isn't produced by the same ion
as the same m/z value peak in pentane.
If you remember, the m/z = 57 peak in pentane was produced by
[CH3CH2CH2CH2]+. If you look at the structure of pentan-3-one, it's
impossible to get that particular fragment from it.
Work along the molecule mentally chopping bits off until you come
up with something that adds up to 57. With a small amount of
patience, you'll eventually find [CH3CH2CO]+ - which is produced by
this fragmentation:

You would get exactly the same products whichever side of the CO

group you split the molecular ion.


The m/z = 29 peak is produced by the ethyl ion - which once again
could be formed by splitting the molecular ion either side of the CO
group.

Peak heights and the stability of ions


The more stable an ion is, the more likely it is to form. The more of
a particular sort of ion that's formed, the higher its peak height will
be. We'll look at two common examples of this.
Examples involving carbocations (carbonium ions)
Important! If you don't know what a carbocation (or carbonium
ion) is, or why the various sorts vary in stability, it's essential that
you follow this link before you go on.
Use the BACK button on your browser to return quickly to this
page.

Summarizing the most important conclusion from the page on


carbocations:

Order of stability of carbocations


primary < secondary < tertiary

Note: The symbol "<" means "is less than". So what this is
saying is that primary ions are less stable than secondary ones
which in turn are less stable than tertiary ones.

Applying the logic of this to fragmentation patterns, it means that a


split which produces a secondary carbocation is going to be more
successful than one producing a primary one. A split producing a
tertiary carbocation will be more successful still.
Let's look at the mass spectrum of 2-methylbutane. 2-methylbutane
is an isomer of pentane - isomers are molecules with the same
molecular formula, but a different spatial arrangement of the atoms.

Look first at the very strong peak at m/z = 43. This is caused by a
different ion than the corresponding peak in the pentane mass
spectrum. This peak in 2-methylbutane is caused by:

The ion formed is a secondary carbocation - it has two alkyl groups


attached to the carbon with the positive charge. As such, it is
relatively stable.
The peak at m/z = 57 is much taller than the corresponding line in
pentane. Again a secondary carbocation is formed - this time, by:

You would get the same ion, of course, if the left-hand CH 3group
broke off instead of the bottom one as we've drawn it.
In these two spectra, this is probably the most dramatic example of
the extra stability of a secondary carbocation.

Examples involving acylium ions, [RCO] +


Ions with the positive charge on the carbon of a carbonyl group,
C=O, are also relatively stable. This is fairly clearly seen in the
mass spectra of ketones like pentan-3-one.

The base peak, at m/z=57, is due to the [CH3CH2CO]+ ion. We've


already discussed the fragmentation that produces this.
Note: There are lots of other examples of positive ions with extra
stability and which are produced in large numbers in a mass
spectrometer as a result. Without making this article even longer
than it already is, it's impossible to cover every possible case.
Check past exam papers to find out whether you are likely to
need to know about other possibilities. If you haven't got past
papers, follow the link on the syllabuses page to find out how to
get hold of them.

Using mass spectra to distinguish between compounds


Suppose you had to suggest a way of distinguishing between
pentan-2-one and pentan-3-one using their mass spectra.
pentan-2-one

CH3COCH2CH2CH3

pentan-3-one

CH3CH2COCH2CH3

Each of these is likely to split to produce ions with a positive charge

on the CO group.
In the pentan-2-one case, there are two different ions like this:

[CH3CO]+

[COCH2CH2CH3]+

That would give you strong lines at m/z = 43 and 71.


With pentan-3-one, you would only get one ion of this kind:

[CH3CH2CO]+

In that case, you would get a strong line at 57.


You don't need to worry about the other lines in the spectra - the
43, 57 and 71 lines give you plenty of difference between the two.
The 43 and 71 lines are missing from the pentan-3-one spectrum,
and the 57 line is missing from the pentan-2-one one.
Note: Don't confuse the line at m/z = 58 in the pentan-2-one
spectrum. That's due to a complicated rearrangement which you
couldn't possibly predict at A'level.

The two spectra look like this:

Computer matching of mass spectra


As you've seen, the mass spectrum of even very similar organic
compounds will be quite different because of the different
fragmentations that can occur. Provided you have a computer data
base of mass spectra, any unkown spectrum can be computer
analysed and simply matched against the data base.

Questions to test your understanding


If this is the first set of questions you have done, please read theintroductory
page before you start. You will need to use the BACK BUTTON on your
browser to come back here afterwards.
questions on fragmentation patterns
answers

Where would you like to go now?


To the mass spectrometry menu . . .
To the instrumental analysis menu . . .
To Main Menu . . .

EI-MassSpectra of Assorted Organic Compounds


The mass apectra of three different saturated hydrocarbons are displayed below. Two
are isomeric hexanes and the third is cyclohexane. Comments regarding the
fragmentation patterns are presented in the box to the right of each spectrum. Ions are
sometimes characterized by loss of a specific neutral fragment from the molecular ion.
For example, a M-15 ion is identified as loss of a methyl group. Odd-electron ions,
including the molecular ion, are colored orange when marked. Even-electron ions are
colored magenta. The "Toggle Examples" button at the bottom will display a different set
of spectra in which the influence of a particular functional group may be examined.
Repeated clicking of this button will cycle through fifteeen spectra. In each example the
molecular ion is designated by M +.

1. These three examples are hydrocarbons having no functional groups.


--------------------------------------------------------------------------Hexane shows the same fragmentation pattern as other unbranched alkanes. Thus, alkyl
carbocations at m/z=15, 29, 43 and 57 Da provide the dominant peaks in the spectrum.
The m/z=57 butyl cation (M-29) is the base peak, and the m/z=43 and 29 ions are also
abundant.

2. Chain branching clearly influences the fragmentation of this isomeric hexane. The
molecular ion at m/z=86 is weaker than that for hexane itself and the M-15 ion at m/z=71
is stronger. The m/z=57 ion is almost absent (try to find a simple cleavage that gives a
butyl group). An isopropyl cation (m/z=43) is very strong, and the corresponding propene
radical-cation at m/z=42 (colored orange), produced by loss of propane, gives the base
peak.
3. By having the six carbons of hexane closed to a ring, the fragmentation is profoundly
changed. To begin with, the molecular ion at m/z=84 is much stronger than the
corresponding ions in the previous acyclic compounds. The base peak at m/z=56 is
produced by loss of ethene, so it is an odd-electron ion (colored orange). The alkenyl
cations at m/z=41 & 27 are stronger than the corresponding alkyl cations (m/z=43 & 29).
The loss of methyl (m/z=69), and a corresponding small m/z=15 ion obviously require
some hydrogen rearrangements.

4. These three examples are carbonyl compounds.


---------------------------------------------------------------------------

Pentanal displays a set of ions associated with the alkyl chain (e.g. m/z=57, 43, 41, 29 &
27). The molecular ion at m/z=86 is very weak, as is the M-1 ion at m/z=85 (aldehydes
generally show fragment ions involving loss of groups attached to the carbonyl function).
Thus, the m/z=29 ion is probably composed of both ethyl and HCO cations. Oddelectron
5. The molecular ion at m/z=86 is more abundant than in the previous aldehyde
spectrum. The directive effect of the carbonyl group on fragmentation is also apparent
here. Loss of carbonyl substituents by alpha-fragmentation include: loss of methyl (M-15
at m/z=71) and loss of a propyl radical (M-43 at m/z=43). These comprise the most
prominent fragment ions.
6. The molecular ion at m/z=86 is about as strong as in 2-pentanone. Because of the
symmetry, ethyl groups are the only carbonyl substituents that can be lost by an alphafragmentation. The strongest fragment ions are found at m/z=57 (loss of an ethyl group)
and m/z=29 (an ethyl cation).

7. Alcohols and Sulfides

---------------------------------------------------------------------------In 1-pentanol the hydroxyl group is at the end of the five-carbon chain. There are two
significant odd-electron fragment ions, one at m/z=70 (loss of water), and the other at
m/z=42 (loss of water and ethene). The fragment ion at m/z=55 is probably due to a
methyl radical loss from the m/z=70 ion. The m/z=31 ion may be a protonated
formaldehyde ion, formed by alpha-fragmentation.
8. 3-Pentanol shows three significant fragment ions. Alpha-fragmentation (loss of an
ethyl radical) forms the m/z=59 base peak. Loss of water from this gives a m/z=41
fragment, and loss of ethene from m/z=59 gives a m/z=31 fragment.
9. The molecular ion (m/z=90) is strong, and the presence of sulfur is indicated by a
larger than usual M+2 (m/z=92) peak. This is true for the m/z=75 & 47 peaks as well.
Loss of methyl and ethyl radicals generate ions at m/z=75 & 61. The odd-electron
rearrangement ion at m/z=62 results from loss of ethene. Finally, m/z=47 may be formed
from m/z=62 by loss of a methyl radical.

10.

Amines

------------------------------------------------------------------------The mass spectrum of1-aminopentane is remarkably simple, thanks to the directive


influence of nitrogen. Alpha-fragmentation generates the m/z=30 even-electron cation,
which is the only significant fragment ion. The molecular ion (m/z=87) is rather weak.
11. The mass spectrum of 3-aminopentane is only slightly more complex than is isomer
above. The molecular ion is very weak, and alpha-fragmentation again provides the
base peak (m/z=58). Loss of ammonia from this ion generates the m/z=41 ion.
12. The cyclic secondary amine, piperidine, has a more complex mass spectrum. The
molecular ion at m/z=85 is relatively strong. Alpha-fragmentation of a hydrogen gives the
M-1 base peak. Because of the ring, alpha-fragmentation of a carbon group does not
result in a change of mass. This ring-opened cation-radical may then lose an ethyl
radical or ethene to give respectively m/z=56 & 57 ions. Loss of an allyl radical from the
ring-opened molecular ion would produce the m/z=44 ion.

13. Esters and Amides


-------------------------------------------------------------------------

The molecular ion in the mass spectrum of ethyl acetate is rather weak. The base peak
results from an alpha-fragmentation of ethoxyl radical to give an m/z=43 ion. It is
interesting that the alternative alpha-cleavage to an M-15 ion is very weak. The loss of
water to generate the odd-electron ion at m/z=70 is curious. Small methyl and ethyl ions
are found at m/z=15 & 29.
14. The isomeric ester, methyl propanoate, has a more abundant molecular ion than
ethyl acetate. The alpha fragmentation of methoxyl radical generates the strong m/z=57
ion. Alpha-fragmentation of the ethyl group leads to both m/z=59 & 29. A smaller methyl
peak is also seen.
15. The stability of dimethylformamide (DMF) is evident in the abundance of its
molecular ion (m/z=73), which is also the base peak. A small M-15 peak is observed, but
the second most abundant ion is loss of HCO by an alpha-cleavage (m/z=44). Other
ions at m/z=42, 30 & 28 are probably derived from the m/z=44 ion.

End of this supplementary topic

Common Fragment Ions and Neutral Fragments


m/z
15 Da
17
18
19
26
27
28
29
30
31
33
34

Common Small Ions


composition
CH3
OH
H2O
H3O, F
C2H2, CN
C2H3
C2H4, CO, H2CN
C2H5, CHO
CH2NH2
CH3O
SH, CH2F
H2S

Common Neutral Fragments


mass loss
composition
1 Da
H
15
CH3
17
OH
18
H2O
19
F
20
HF
27
C2H3, HCN
28
C2H4, CO
30
CH2O
31
CH3O
32
CH4O, S
33
CH3 + H2O, HS

35(37)
36(38)
39
41
42
43
44
46
56
57
60
79(81)
80(82)
91
127
128

Cl
HCl
C3H3
C3H5, C2H3N
C3H6, C2H2O, C2H4N
C3H7, CH3CO
C2H4O
NO2
C4H8
C4H9
CH4CO2
Br
HBr
C7H7
I
HI

33
35(37)
36(38)
42
43
44
45
55
57
59
60
64
79(81)
80(82)
127
128

H2S
Cl
HCl
C3H6, C2H2O, C2H4N
C3H7, CH3CO
CO2O, CONH2
C2H5O
C4H7
C4H9
C2H3O2
C2H4O2
SO2
Br
HBr
I
HI

End of this supplementary topic

Rearangement Mechanisms in Fragmentation

4-nonanone

The odd-electron fragment ions at


m/z = 86 and 58 are the result of a
McLafferty rearrangement, involving
the larger alkyl chain, and a
subsequent loss of ethene (the
"double-McLafferty" rearrangement).
Alpha-cleavage leads to the m/z = 99,
71 and 43 ions. The charge is
apparently distributed over both
fragments.

butylpentanoate

Alpha-cleavage gives ions at m/z=57


& 85 Da. The McLafferty
rearrangement on the acid side
generates a m/z=116 ion. Subsequent
rearrangement on the alcohol side
generates m/z=60 and 56 ions. The
m/z=103 ion is probably
C4H9CO2H2(+).

5-methyl-5-hexen-3-ol
The molecular ion (m/z=114 Da) is
not observed under electron impact
ionization conditions. The highest
mass ion (m/z=85) is due to an alphacleavage of ethyl; the other alphacleavage generates m/z=59. The
rearrangement cleavage shown here
generates the m/z=56 ion.

4,4-dimethylcyclohexene
The loss of a methyl radical generates
the base peak at m/z=95 Da. The
m/z=81 & 67 ions are smaller
homologues of this ion (14 mass
units less). Cyclohexene compounds
undergo a retro-Diel-Alder
rearrangement to give diene and
alkene fragments. The charge may
reside on either fragment, with the
larger usually predominating. In this
case both ions are relatively strong
(m/z=54 & 56).

Section 4.1: Introduction to molecular


spectroscopy

4.1A: The electromagnetic spectrum


Electromagnetic radiation, as you may recall from a previous chemistry or physics class, is composed of
electrical and magnetic waves which oscillate on perpendicular planes. Visible light is electromagnetic radiation.
So are the gamma rays that are emitted by spent nuclear fuel, the x-rays that a doctor uses to visualize your
bones, the ultraviolet light that causes a painful sunburn when you forget to apply sun block, the infrared light that
the army uses in night-vision goggles, the microwaves that you use to heat up your frozen burritos, and the radiofrequency waves that bring music to anybody who is old-fashioned enough to still listen to FM or AM radio.
Just like ocean waves, electromagnetic waves travel in a defined direction. While the speed of ocean waves can
vary, however, the speed of electromagnetic waves commonly referred to as the speed of light is essentially a
constant, approximately 300 million meters per second. This is true whether we are talking about gamma
radiation or visible light. Obviously, there is a big difference between these two types of waves we are
surrounded by the latter for more than half of our time on earth, whereas we hopefully never become exposed to
the former to any significant degree. The different properties of the various types of electromagnetic radiation are
due to differences in their wavelengths, and the corresponding differences in their energies: shorter wavelengths
correspond to higher energy.

High-energy radiation (such as gamma- and x-rays) is composed of very short waves as short as 10 -16 meter
from crest to crest. Longer waves are far less energetic, and thus are less dangerous to living things. Visible
light waves are in the range of 400 700 nm (nanometers, or 10 -9 m), while radio waves can be several hundred
meters in length.
The notion that electromagnetic radiation contains a quantifiable amount of energy can perhaps be better
understood if we talk about light as a stream of particles, called photons, rather than as a wave. (Recall the
concept known as wave-particle duality: at the quantum level, wave behavior and particle behavior become
indistinguishable, and very small particles have an observable wavelength). If we describe light as a stream of
photons, the energy of a particular wavelength can be expressed as:
E = hc/
where E is energy in kcal/mol, (the Greek letter lambda) is wavelength in meters, c is 3.00 x 108 m/s (the speed
of light), and h is 9.537 x 10-14 kcalsmol-1, a number known as Plancks constant.
Because electromagnetic radiation travels at a constant speed, each wavelength corresponds to a given
frequency, which is the number of times per second that a crest passes a given point. Longer waves have lower
frequencies, and shorter waves have higher frequencies. Frequency is commonly reported in hertz (Hz),
meaning cycles per second, or waves per second. The standard unit for frequency is s-1.

When talking about electromagnetic waves, we can refer either to wavelength or to frequency - the two values
are interconverted using the simple expression:
= c
where (the Greek letter nu) is frequency in s-1. Visible red light with a wavelength of 700 nm, for example, has
a frequency of 4.29 x 1014Hz, and an energy of 40.9 kcal per mole of photons.
The full range of electromagnetic radiation wavelengths is referred to as the electromagnetic spectrum.

Notice in the figure above that visible light takes up just a narrow band of the full spectrum. White light from the
sun or a light bulb is a mixture of all of the visible wavelengths. You see the visible region of the electromagnetic
spectrum divided into its different wavelengths every time you see a rainbow: violet light has the shortest
wavelength, and red light has the longest.

Example
Exercise 4.1: Visible light has a wavelength range of about 400-700 nm. What is the corresponding frequency
range? What is the corresponding energy range, in kcal/mol of photons?
Solution

4.1B: Molecular spectroscopy the basic


idea
In a spectroscopy experiment, electromagnetic radiation of a specified range of wavelengths is allowed to pass
through a sample containing a compound of interest. The sample molecules absorb energy from some of the
wavelengths, and as a result jump from a low energy ground state to some higher energy excited state. Other
wavelengths are not absorbed by the sample molecule, so they pass on through. A detector on the other side of
the sample records which wavelengths were absorbed, and to what extent they were absorbed.

Here is the key to molecular spectroscopy: a given molecule will specifically absorb only those wavelengths
which have energies that correspond to the energy difference of the transition that is occurring. Thus, if the
transition involves the molecule jumping from ground state A to excited state B, with an energy difference of E,
the molecule will specifically absorb radiation with wavelength that corresponds to E, while allowing other
wavelengths to pass through unabsorbed.

By observing which wavelengths a molecule absorbs, and to what extent it absorbs them, we can gain
information about the nature of the energetic transitions that a molecule is able to undergo, and thus information
about its structure.
These generalized ideas may all sound quite confusing at this point, but things will become much clearer as we
begin to discuss specific examples.

Section 4.2: Infrared spectroscopy


Covalent bonds in organic molecules are not rigid sticks rather, they behave more like springs. At room
temperature, organic molecules are always in motion, as their bonds stretch, bend, and twist. These complex
vibrations can be broken down mathematically into individualvibrational modes, a few of which are illustrated
below.

The energy of molecular vibration is quantized rather than continuous, meaning that a molecule can only stretch
and bend at certain 'allowed' frequencies. If a molecule is exposed to electromagnetic radiation that matches the
frequency of one of its vibrational modes, it will in most cases absorb energy from the radiation and jump to a
higher vibrational energy state - what this means is that the amplitude of the vibration will increase, but the
vibrational frequency will remain the same. The difference in energy between the two vibrational states is equal
to the energy associated with the wavelength of radiation that was absorbed. It turns out that it is
the infrared region of the electromagnetic spectrum which contains frequencies corresponding to the vibrational
frequencies of organic bonds.
Let's take 2-hexanone as an example. Picture the carbonyl bond of the ketone group as a spring. This spring is
constantly bouncing back and forth, stretching and compressing, pushing the carbon and oxygen atoms further
apart and then pulling them together. This is thestretching mode of the carbonyl bond. In the space of one
second, the spring 'bounces' back and forth 5.15 x 1013 times - in other words, the ground-state frequency of
carbonyl stretching for a the ketone group is about 5.15 x 1013 Hz.
If our ketone sample is irradiated with infrared light, the carbonyl bond will specifically absorb light with this same
frequency, which by equations 4.1 and 4.2 corresponds to a wavelength of 5.83 x 10-6 m and an energy of 4.91
kcal/mol. When the carbonyl bond absorbs this energy, it jumps up to an excited vibrational state.

The value of E - the energy difference between the low energy (ground) and high energy (excited) vibrational
states - is equal to 4.91 kcal/mol, the same as the energy associated with the absorbed light frequency. The
molecule does not remain in its excited vibrational state for very long, but quickly releases energy to the
surrounding environment in form of heat, and returns to the ground state.
With an instrument called an infrared spectrophotometer, we can 'see' this vibrational transition. In the
spectrophotometer, infrared light with frequencies ranging from about 10 13 to 1014 Hz is passed though our
sample of cyclohexane. Most frequencies pass right through the sample and are recorded by a detector on the
other side.

Our 5.15 x 1013 Hz carbonyl stretching frequency, however, is absorbed by the 2-hexanone sample, and so the
detector records that the intensity of this frequency, after having passed through the sample, is something less
than 100% of its initial intensity.
The vibrations of a 2-hexanone molecule are not, of course, limited to the simple stretching of the carbonyl bond.
The various carbon-carbon bonds also stretch and bend, as do the carbon-hydrogen bonds, and all of these
vibrational modes also absorb different frequencies of infrared light.

The power of infrared spectroscopy arises from the observation that different functional groups have different
characteristic absorption frequencies. The carbonyl bond in a ketone, as we saw with our 2-hexanone example,
typically absorbs in the range of 5.11 - 5.18 x 1013Hz, depending on the molecule. The carbon-carbon triple
bond of an alkyne, on the other hand, absorbs in the range 6.30 - 6.80 x 1013Hz. The technique is therefore very
useful as a means of identifying which functional groups are present in a molecule of interest. If we pass infrared
light through an unknown sample and find that it absorbs in the carbonyl frequency range but not in the alkyne
range, we can infer that the molecule contains a carbonyl group but not an alkyne.
Some bonds absorb infrared light more strongly than others, and some bonds do not absorb at all. In order for a
vibrational mode to absorb infrared light, it must result in a periodic change in the dipole moment of the
molecule. Such vibrations are said to be infrared active. In general, the greater the polarity of the bond, the
stronger its IR absorption. The carbonyl bond is very polar, and absorbs very strongly. The carbon-carbon triple
bond in most alkynes, in contrast, is much less polar, and thus a stretching vibration does not result in a large
change in the overall dipole moment of the molecule. Alkyne groups absorb rather weakly compared to
carbonyls.
Some kinds of vibrations are infrared inactive. The stretching vibrations of completely symmetrical double and
triple bonds, for example, do not result in a change in dipole moment, and therefore do not result in any
absorption of light (but other bonds and vibrational modes in these molecules do absorb IR light).

Now, let's look at some actual output from IR spectroscopy experiments. Below is the IR spectrum for 2hexanone.

There are a number of things that need to be explained in order for you to understand what it is that we are
looking at. On the horizontal axis we see IR wavelengths expressed in terms of a unit called wavenumber (cm1
), which tells us how many waves fit into one centimeter. On the vertical axis we see % transmittance, which
tells us how strongly light was absorbed at each frequency (100% transmittance means no absorption occurred
at that frequency). The solid line traces the values of % transmittance for every wavelength the peaks (which
are actually pointing down) show regions of strong absorption. For some reason, it is typical in IR spectroscopy
to report wavenumber values rather than wavelength (in meters) or frequency (in Hz). The upside down vertical
axis, with absorbance peaks pointing down rather than up, is also a curious convention in IR spectroscopy. We
wouldnt want to make things too easy for you!

Example

Exercise 4.2: Express the wavenumber value of 3000 cm-1 in terms of wavelength (in meter units).

Solution

The key absorption peak in this spectrum is that from the carbonyl double bond, at 1716 cm -1 (corresponding to a
wavelength of 5.86 mm, a frequency of 5.15 x 10 13 Hz, and a E value of 4.91 kcal/mol). Notice how strong this
peak is, relative to the others on the spectrum: a strong peak in the 1650-1750 cm -1 region is a dead giveaway
for the presence of a carbonyl group. Within that range, carboxylic acids, esters, ketones, and aldehydes tend to
absorb in the shorter wavelength end (1700-1750 cm-1), while conjugated unsaturated ketones and amides tend
to absorb on the longer wavelength end (1650-1700 cm-1).
The jagged peak at approximately 2900-3000 cm -1 is characteristic of tetrahedral carbon-hydrogen bonds. This
peak is not terribly useful, as just about every organic molecule that you will have occasion to analyze has these
bonds. Nevertheless, it can serve as a familiar reference point to orient yourself in a spectrum.
You will notice that there are many additional peaks in this spectrum in the longer-wavelength 400 -1400 cm 1
region. This part of the spectrum is called the fingerprint region. While it is usually very difficult to pick out any
specific functional group identifications from this region, it does, nevertheless, contain valuable information. The
reason for this is suggested by the name: just like a human fingerprint, the pattern of absorbance peaks in the
fingerprint region is unique to every molecule, meaning that the data from an unknown sample can be compared
to the IR spectra of known standards in order to make a positive identification. In the mid-1990's, for example,
several paintings were identified as forgeries because scientists were able to identify the IR footprint region of red
and yellow pigment compounds that would not have been available to the artist who supposedly created the
painting (for more details see Chemical and Engineering News, Sept 10, 2007, p. 28).
Now, lets take a look at the IR spectrum for 1-hexanol.

As you can see, the carbonyl peak is gone, and in its place is a very broad mountain centered at about 3400 cm 1
. This signal is characteristic of the O-H stretching mode of alcohols, and is a dead giveaway for the presence of
an alcohol group. The breadth of this signal is a consequence of hydrogen bonding between molecules.
In the spectrum of octanoic acid we see, as expected, the characteristic carbonyl peak, this time at 1709 cm -1.

We also see a low, broad absorbance band that looks like an alcohol, except that it is displaced slightly to the
right (long-wavelength) side of the spectrum, causing it to overlap to some degree with the C-H region. This is
the characteristic carboxylic acid O-H single bond stretching absorbance.
The spectrum for 1-octene shows two peaks that are characteristic of alkenes: the one at 1642 cm -1 is due to
stretching of the carbon-carbon double bond, and the one at 3079 cm-1 is due to stretching of the s bond
between the alkene carbons and their attached hydrogens.

Alkynes have characteristic IR absorbance peaks in the range of 2100-2250 cm -1 due to stretching of the carboncarbon triple bond, and terminal alkenes can be identified by their absorbance at about 3300 cm-1, due to
stretching of the bond between the sp-hybridized carbon and the terminal hydrogen.
It is possible to identify other functional groups such as amines and ethers, but the characteristic peaks for these
groups are considerably more subtle and/or variable, and often are overlapped with peaks from the fingerprint
region. For this reason, we will limit our discussion here to the most easily recognized functional groups, which
are summarized in table 1 in the tables section at the end of the text.
As you can imagine, obtaining an IR spectrum for a compound will not allow us to figure out the complete
structure of even a simple molecule, unless we happen to have a reference spectrum for comparison. In
conjunction with other analytical methods, however, IR spectroscopy can prove to be a very valuable tool, given
the information it provides about the presence or absence of key functional groups. IR can also be a quick and
convenient way for a chemist to check to see if a reaction has proceeded as planned. If we were to run a
reaction in which we wished to convert cyclohexanone to cyclohexanol, for example, a quick comparison of the
IR spectra of starting compound and product would tell us if we had successfully converted the ketone group to
an alcohol (this type of reaction is discussed in detail in chapter 16).

Internal Links

Virtual Textbook of OChem: IR spectroscopy

Section 4.3: Ultraviolet and visible spectroscopy


While interaction with infrared light causes molecules to undergo vibrational transitions, the shorter wavelength,
higher energy radiation in the UV (200-400 nm) and visible (400-700 nm) range of the electromagnetic spectrum
causes many organic molecules to undergo electronic transitions. What this means is that when the energy
from UV or visible light is absorbed by a molecule, one of its electrons jumps from a lower energy to a higher
energy molecular orbital.

4.3A: Electronic transitions


Lets take as our first example the simple case of molecular hydrogen, H 2. As you may recall from section 2.1A,
the molecular orbital picture for the hydrogen molecule consists of one bonding MO, and a higher energy
antibonding * MO. When the molecule is in the ground state, both electrons are paired in the lower-energy
bonding orbital this is the Highest Occupied Molecular Orbital (HOMO). The antibonding * orbital, in turn, is
the Lowest Unoccupied Molecular Orbital (LUMO).

If the molecule is exposed to light of a wavelength with energy equal to E, the HOMO-LUMO energy gap, this
wavelength will be absorbed and the energy used to bump one of the electrons from the HOMO to the LUMO in
other words, from the to the * orbital. This is referred to as a - * transition. E for this electronic transition
is 258 kcal/mol, corresponding to light with a wavelength of 111 nm.
When a double-bonded molecule such as ethene (common name ethylene) absorbs light, it undergoes a - *
transition. Because - * energy gaps are narrower than - * gaps, ethene absorbs light at 165 nm - a longer
wavelength than molecular hydrogen.

The electronic transitions of both molecular hydrogen and ethene are too energetic to be accurately recorded by
standard UV spectrophotometers, which generally have a range of 220 700 nm. Where UV-vis spectroscopy

becomes useful to most organic and biological chemists is in the study of molecules with conjugated pi systems.
In these groups, the energy gap for -* transitions is smaller than for isolated double bonds, and thus the
wavelength absorbed is longer. Molecules or parts of molecules that absorb light strongly in the UV-vis region
are called chromophores.
Lets revisit the MO picture for 1,3-butadiene, the simplest conjugated system (see section 2.1B). Recall that we
can draw a diagram showing the four pi MOs that result from combining the four 2p z atomic orbitals. The lower
two orbitals are bonding, while the upper two are antibonding.

Comparing this MO picture to that of ethene, our isolated pi-bond example, we see that the HOMO-LUMO energy
gap is indeed smaller for the conjugated system. 1,3-butadiene absorbs UV light with a wavelength of 217 nm.
As conjugated pi systems become larger, the energy gap for a - * transition becomes increasingly narrow, and
the wavelength of light absorbed correspondingly becomes longer. The absorbance due to the - * transition in
1,3,5-hexatriene, for example, occurs at 258 nm, corresponding to a E of 111 kcal/mol.

In molecules with extended pi systems, the HOMO-LUMO energy gap becomes so small that absorption occurs
in the visible rather then the UV region of the electromagnetic spectrum. Beta-carotene, with its system of 11
conjugated double bonds, absorbs light with wavelengths in the blue region of the visible spectrum while
allowing other visible wavelengths mainly those in the red-yellow region - to be transmitted. This is why carrots
are orange.

The conjugated pi system in 4-methyl-3-penten-2-one gives rise to a strong UV absorbance at 236 nm due to
a - * transition. However, this molecule also absorbs at 314 nm. This second absorbance is due to the
transition of a non-bonding (lone pair) electron on the oxygen up to a * antibonding MO:

This is referred to as an n - * transition. The nonbonding (n) MOs are higher in energy than the highest
bonding p orbitals, so the energy gap for an n - * transition is smaller that that of a - * transition and thus the
n - * peak is at a longer wavelength. In general, n - * transitions are weaker (less light absorbed) than those
due to - * transitions.

Example
Exercise 4.3: How large is the - * transition in 4-methyl-3-penten-2-one?
Exercise 4.4: Which of the following molecules would you expect absorb at a longer wavelength in the UV region
of the electromagnetic spectrum? Explain your answer.

Solution

4.3B: Looking at UV-vis spectra


We have been talking in general terms about how molecules absorb UV and visible light now let's look at some
actual examples of data from a UV-vis absorbance spectrophotometer. The basic setup is the same as for IR
spectroscopy: radiation with a range of wavelengths is directed through a sample of interest, and a detector
records which wavelengths were absorbed and to what extent the absorption occurred. Below is the absorbance
spectrum of an important biological molecule called nicotinamide adenine dinucleotide, abbreviated NAD+ (we'll
learn what it does in section 16.4) This compound absorbs light in the UV range due to the presence of
conjugated pi-bonding systems.

Youll notice that this UV spectrum is much simpler than the IR spectra we saw earlier: this one has only one
peak, although many molecules have more than one. Notice also that the convention in UV-vis spectroscopy is
to show the baseline at the bottom of the graph with the peaks pointing up. Wavelength values on the x-axis are
generally measured in nanometers (nm) rather than in cm-1 as is the convention in IR spectroscopy.
Peaks in UV spectra tend to be quite broad, often spanning well over 20 nm at half-maximal height. Typically,
there are two things that we look for and record from a UV-Vis spectrum.. The first is max, which is the
wavelength at maximal light absorbance. As you can see, NAD+has max, = 260 nm. We also want to record how
much light is absorbed at max. Here we use a unitless number called absorbance, abbreviated 'A'. This contains
the same information as the 'percent transmittance' number used in IR spectroscopy, just expressed in slightly
different terms. To calculate absorbance at a given wavelength, the computer in the spectrophotometer simply
takes the intensity of light at that wavelength before it passes through the sample (I 0), divides this value by the
intensity of the same wavelength after it passes through the sample (I), then takes the log10 of that number:
A = log I0/I

You can see that the absorbance value at 260 nm (A260) is about 1.0 in this spectrum.

Example
Exercise 4.5: Express A = 1.0 in terms of percent transmittance (%T, the unit usually used in IR spectroscopy
(and sometimes in UV-vis as well).
Solution

Here is the absorbance spectrum of the common food coloring Red #3:

Here, we see that the extended system of conjugated pi bonds causes the molecule to absorb light in the visible
range. Because the max of 524 nm falls within the green region of the spectrum, the compound appears red to
our eyes.
Now, take a look at the spectrum of another food coloring, Blue #1:

Here, maximum absorbance is at 630 nm, in the orange range of the visible spectrum, and the compound
appears blue.

4.3C: Applications of UV spectroscopy in


organic and biological chemistry
UV-vis spectroscopy has many different applications in organic and biological chemistry. One of the most basic
of these applications is the use of the Beer - Lambert Law to determine the concentration of a chromophore.
You most likely have performed a Beer Lambert experiment in a previous chemistry lab. The law is simply an
application of the observation that, within certain ranges, the absorbance of a chromophore at a given
wavelength varies in a linear fashion with its concentration: the higher the concentration of the molecule, the
greater its absorbance. If we divide the observed value of A at max by the concentration of the sample (c, in
mol/L), we obtain the molar absorptivity, or extinction coefficient (), which is a characteristic value for a given
compound.

= A/c
The absorbance will also depend, of course, on the path length - in other words, the distance that the beam of
light travels though the sample. In most cases, sample holders are designed so that the path length is equal to 1
cm, so the units for molar absorptivity are mol * L-1cm-1. If we look up the value of e for our compound at max, and
we measure absorbance at this wavelength, we can easily calculate the concentration of our sample.

As an

example, for NAD+ the literature value of

at 260 nm is 18,000 mol * L

-1

cm-1. In our NAD+ spectrum we observed

A260 = 1.0, so using equation 4.4 and solving for concentration we find that our sample is 5.6 x 10-5 M.

Example
Exercise 4.6: The literature value of for 1,3-pentadiene in hexane is 26,000 mol * L -1cm-1 at its maximum
absorbance at 224 nm. You prepare a sample and take a UV spectrum, finding that A 224 = 0.850. What is the
concentration of your sample?
Solution

The bases of DNA and RNA are good chromophores:

Biochemists and molecular biologists often determine the concentration of a DNA sample by assuming an
average value of = 0.020 ng-1mL for double-stranded DNA at its max of 260 nm (notice that concentration in
this application is expressed in mass/volume rather than molarity: ng/mL is often a convenient unit for DNA
concentration when doing molecular biology).

Example
Exercise 4.7: 50 mL of an aqueous sample of double stranded DNA is dissolved in 950 mL of water. This diluted
solution has a maximal absorbance of 0.326 at 260 nm. What is the concentration of the original (more
concentrated) DNA sample, expressed in mg/mL?
Solution

Because the extinction coefficient of double stranded DNA is slightly lower than that of single stranded DNA, we
can use UV spectroscopy to monitor a process known as DNA melting. If a short stretch of double stranded
DNA is gradually heated up, it will begin to melt, or break apart, as the temperature increases (recall that two
strands of DNA are held together by a specific pattern of hydrogen bonds formed by base-pairing).

As melting proceeds, the absorbance value for the sample increases, eventually reaching a high plateau as all of
the double-stranded DNA breaks apart, or melts. The mid-point of this process, called the melting temperature,
provides a good indication of how tightly the two strands of DNA are able to bind to each other.
In section 16.8 we will see how the Beer - Lambert Law and UV spectroscopy provides us with a convenient way
to follow the progress of many different enzymatic redox (oxidation-reduction) reactions. In biochemistry,
oxidation of an organic molecule often occurs concurrently with reduction of nicotinamide adenine dinucleotide
(NAD+, the compound whose spectrum we saw earlier in this section) to NADH:

Both NAD+ and NADH absorb at 260 nm. However NADH, unlike NAD+, has a second absorbance band
with max = 340 nm and = 6290 mol L-1cm-1. The figure below shows the spectra of both compounds
superimposed, with the NADH spectrum offset slightly on the y-axis:

By monitoring the absorbance of a reaction mixture at 340 nm, we can 'watch' NADH being formed as the
reaction proceeds, and calculate the rate of the reaction.
UV spectroscopy is also very useful in the study of proteins. Proteins absorb light in the UV range due to the
presence of the aromatic amino acids tryptophan, phenylalanine, and tyrosine, all of which are chromophores.

Biochemists frequently use UV spectroscopy to study conformational changes in proteins - how they change
shape in response to different conditions. When a protein undergoes a conformational shift (partial unfolding, for
example), the resulting change in the environment around an aromatic amino acid chromophore can cause its
UV spectrum to be altered.

Section 4.4: Mass Spectrometry


Our third and final analytical technique for discussion in this chapter does not fall under the definition of
spectroscopy, as it does not involve the absorbance of light by a molecule. In mass spectrometry (MS), we are
interested in the mass - and therefore the molecular weight - of our compound of interest, and often the mass of
fragments that are produced when the molecule is caused to break apart.

4.4A: The basics of a mass spectrometry


There are many different types of MS instruments, but they all have the same three essential components. First,
there is an ionization source, where the molecule is given a positive electrical charge, either by removing an
electron or by adding a proton. Depending on the ionization method used, the ionized molecule may or may not
break apart into a population of smaller fragments. In the figure below, some of the sample molecules remain
whole, while others fragment into smaller pieces.
Next in line there is a mass analyzer, where the cationic fragments are separated according to their mass.

Finally, there is a detector, which detects and quantifies the separated ions.
One of the more common types of MS techniques used in the organic laboratory is electron ionization. In the
ionization source, the sample molecule is bombarded by a high-energy electron beam, which has the effect of
knocking a valence electron off of the molecule to form aradical cation. Because a great deal of energy is
transferred by this bombardment process, the radical cation quickly begins to break up into smaller fragments,
some of which are positively charged and some of which are neutral. The neutral fragments are either adsorbed
onto the walls of the chamber or are removed by a vacuum source. In the mass analyzer component, the
positively charged fragments and any remaining unfragmented molecular ions are accelerated down a tube by
an electric field.

This tube is curved, and the ions are deflected by a strong magnetic field. Ions of different mass to charge (m/z)
ratios are deflected to a different extent, resulting in a sorting of ions by mass (virtually all ions have charges of z
= +1, so sorting by the mass to charge ratio is the same thing as sorting by mass). A detector at the end of the
curved flight tube records and quantifies the sorted ions.

4.4B: Looking at mass spectra


Below is typical output for an electron-ionization MS experiment (MS data in the section is derived from
the Spectral Database for Organic Compounds, a free, web-based service provided by AIST in Japan.

The sample is acetone. On the horizontal axis is the value for m/z (as we stated above, the charge z is almost
always +1, so in practice this is the same as mass). On the vertical axis is the relative abundance of each ion
detected. On this scale, the most abundant ion, called thebase peak, is set to 100%, and all other peaks are
recorded relative to this value. For acetone, the base peak is at m/z = 43 - we will discuss the formation of this
fragment a bit later. The molecular weight of acetone is 58, so we can identify the peak at m/z = 58 as that
corresponding to the molecular ion peak, or parent peak. Notice that there is a small peak at m/z = 59: this is
referred to as the M+1 peak. How can there be an ion that has a greater mass than the molecular ion? Simple:
a small fraction - about 1.1% - of all carbon atoms in nature are actually the 13C rather than the 12C isotope.
The 13C isotope is, of course, heavier than 12C by 1 mass unit. In addition, about 0.015% of all hydrogen atoms
are actually deuterium, the 2H isotope. So the M+1 peak represents those few acetone molecules in the sample
which contained either a 13C or 2H.
Molecules with lots of oxygen atoms sometimes show a small M+2 peak (2 m/z units greater than the parent
peak) in their mass spectra, due to the presence of a small amount of 18O (the most abundant isotope of oxygen
is 16O). Because there are two abundant isotopes of both chlorine (about 75% 35Cl and 25% 37Cl) and bromine
(about 50% 79Br and 50% 81Br), chlorinated and brominated compounds have very large and recognizable M+2
peaks. Fragments containing both isotopes of Br can be seen in the mass spectrum of ethyl bromide:

Much of the utility in electron-ionization MS comes from the fact that the radical cations generated in the electronbombardment process tend to fragment in predictable ways. Detailed analysis of the typical fragmentation
patterns of different functional groups is beyond the scope of this text, but it is worthwhile to see a few
representative examples, even if we dont attempt to understand the exact process by which the fragmentation
occurs. We saw, for example, that the base peak in the mass spectrum of acetone is m/z = 43. This is the result
of cleavage at the alpha position - in other words, at the carbon-carbon bond adjacent to the carbonyl. Alpha
cleavage results in the formation of an acylium ion (which accounts for the base peak at m/z = 43) and a methyl
radical, which is neutral and therefore not detected.

After the parent peak and the base peak, the next largest peak, at a relative abundance of 23%, is at m/z = 15.
This, as you might expect, is the result of formation of a methyl cation, in addition to an acyl radical (which is
neutral and not detected).

A common fragmentation pattern for larger carbonyl compounds is called the McLafferty rearrangement:

The mass spectrum of 2-hexanone shows a 'McLafferty fragment' at m/z = 58, while the propene fragment is not
observed because it is a neutral species (remember, only cationic fragments are observed in MS). The base peak
in this spectrum is again an acylium ion.

When alcohols are subjected to electron ionization MS, the molecular ion is highly unstable and thus a parent
peak is often not detected. Often the base peak is from an oxonium ion.

Other functional groups have predictable fragmentation patterns as well. By carefully analyzing the fragmentation
information that a mass spectrum provides, a knowledgeable spectrometrist can often put the puzzle together
and make some very confident predictions about the structure of the starting sample.

Example

Exercise 4.8: The mass spectrum of an aldehyde gives prominent peaks at m/z = 59 (12%, highest value
of m/z in the spectrum), 58 (85%), and 29 (100%), as well as others. Propose a structure, and identify the three
species whose m/z values were listed.
Solution

4.4C: Gas Chromatography - Mass


Spectrometry
Quite often, mass spectrometry is used in conjunction with a separation technique called gas chromatography
(GC). The combined GC-MS procedure is very useful when dealing with a sample that is a mixture of two or
more different compounds, because the various compounds are separated from one another before being
subjected individually to MS analysis. We will not go into the details of gas chromatography here, although if you
are taking an organic laboratory course you might well get a chance to try your hand at GC, and you will almost
certainly be exposed to the conceptually analogous techniques of thin layer and column chromatography. Suffice
it to say that in GC, a very small amount of a liquid sample is vaporized, injected into a long, coiled metal column,
and pushed though the column by helium gas. Along the way, different compounds in the sample stick to the
walls of the column to different extents, and thus travel at different speeds and emerge separately from the end of
the column. In GC-MS, each purified compound is sent directly from the end of GC column into the MS
instrument, so in the end we get a separate mass spectrum for each of the compounds in the original mixed

sample. Because a compound's MS spectrum is a very reliable and reproducible 'fingerprint', we can instruct the
instrument to search an MS database and identify each compound in the sample.
The extremely high sensitivity of modern GC-MS instrumentation makes it possible to detect and identify very
small trace amounts of organic compounds. GC-MS is being used increasingly by environmental chemists to
detect the presence of harmful organic contaminants in food and water samples. Airport security screeners also
use high-speed GC-MS instruments to look for residue from bomb-making chemicals on checked luggage.

4.4D: Mass spectrometry of proteins applications in proteomics


Mass spectrometry has become in recent years an increasingly important tool in the field of proteomics.
Traditionally, protein biochemists tend to study the structure and function of individual proteins. Proteomics
researchers, in contrast, want to learn more about how large numbers of proteins in a living system interact with
each other, and how they respond to changes in the state of the organism. One very important subfield of
proteomics is the search for protein biomarkers for human disease. These can be proteins which are present in
greater quantities in a sick person than in a healthy person, and their detection and identification can provide
medical researchers with valuable information about possible causes or treatments. Detection in a healthy
person of a known biomarker for a disease such as diabetes or cancer could also provide doctors with an early
warning that the patient may be especially susceptible, so that preventive measures could be taken to prevent or
delay onset of the disease.
New developments in MS technology have made it easier to detect and identify proteins that are present in very
small quantities in biological samples. Mass spectrometrists who study proteins often use instrumentation that is
somewhat different from the electron-ionization, magnetic deflection system described earlier. When proteins are
being analyzed, the object is often to ionize the proteins without causing fragmentation, so 'softer' ionization
methods are required. In one such method, called electrospray ionization, the protein sample, in solution, is
sprayed into a tube and the molecules are induced by an electric field to pick up extra protons from the solvent.
Another common 'soft ionization' method is 'matrix-assisted laser desorption ionization' (MALDI). Here, the
protein sample is adsorbed onto a solid matrix, and protonation is achieved with a laser.
Typically, both electrospray ionization and MALDI are used in conjunction with a time-of-flight (TOF) mass
analyzer component.

The ionized proteins are accelerated by an electrode through a column, and separation is achieved because
lighter ions travel at greater velocity than heavier ions with the same overall charge. In this way, the many
proteins in a complex biological sample (such as blood plasma, urine, etc.) can be separated and their individual
masses determined very accurately. Modern protein MS is extremely sensitive very recently, scientists were
even able to obtain a mass spectrum of Tyrannosaurus rex protein from fossilized bone! (Science 2007, 316,
277).
In one recent study, MALDI-TOF mass spectrometry was used to compare fluid samples from lung transplant
recipients who had suffered from tissue rejection to control samples from recipients who had not suffered
rejection. Three peptides (short proteins) were found to be present at elevated levels specifically in the tissue
rejection samples. It is hoped that these peptides might serve as biomarkers to identify patients who are at
increased risk of rejecting their transplanted lungs. (Proteomics 2005, 5, 1705).

Contributors

Organic Chemistry With a Biological Emphasis by Tim Soderberg (University of Minnesota, Morris)

Chapter 4 Solutions
In-chapter exercises
E4.1:
Using

= c, we first rearrange to = c/

to solve for frequency.

For light with a wavelength of 400 nm, the frequency is 7.50 x 1014 Hz:

In the same way, we calculate that light with a wavelength of 700 nm has a frequency of 4.29 x 1014 Hz.
To calculate corresponding energies:
Using hc/, we find for light at 400 nm:

Using the same equation, we find that light at 700 nm corresponds to 40.9 kcal/mol.

E4.2:
A wavenumber of 3000 cm-1 means that 3000 waves fit in one cm (0.01m):

We want to find the length of 1 wave, so we divide numerator and denominator by 3000:

So 3000 cm-1 is equivalent to a wavelength of 3.33 mm.

E4.3:
The * transition in 4-methyl-3-penten-2-one is at 236 nm, which corresponds to 121 kcal/mol:

E4.4:
Molecule B has a longer system of conjugated pi bonds, and thus will absorb at a longer wavelength. Notice that
there is an sp3-hybridized carbon in molecule B which isolates two of the pi bonds from the other three.

E4.5:
We use the formula:

recall from your high school algebra that if y = log(x), then x = 10y , so:

. . . so the intensity of light entering the sample (I0) is 10 times the intensity of the light (at a particular wavelength)
that emerges from the sample and is detected (I). Because %T is simply the reciprocal of this ratio multiplied by
100, we find that A = 1.0 corresponds to 10% transmission.

E4.6: Using = A/c, we plug in our values for and A and find that c = 3.27 x 10-5M, or 32.7 mM.

E4.7:
Using = A/c and plugging in the given values for

and A, we

find that c = 16.3 ng/mL. However, this is the

concentration of the diluted solution - the original solution was 20 times higher (50 mL were removed and diluted
to 1000 mL to take the UV measurement). Thus the concentration of the original solution is (20)(16.3) = 326
ng/mL.

E4.8:
The sample is propanal. The peak at m/z = 58 is the molecular ion (parent peak), and the peak at m/z = 29 is the
formyl acylium ion. The M+1 peak at m/z = 59 is a molecular ion in which one of the three carbons is a 13C.

End-of-chapter problems
P4.1:
Without doing any calculation, we can answer the first part of the calculation: electromagnetic waves at 3400 cm1
are shorter than those at 1690 cm-1 (more waves fit into one centimeter) and thus correspond to a higher
frequency.

3400 cm-1 = 2.94 mm = 1.02 x 1014 Hz


1690 cm-1 = 5.92 mm = 5.07 x 1013 Hz

P4.2:
1720 cm-1 corresponds to a wavelength of .01/1720 = 5.81 x 10-6 m, and an energy of 4.92 kcal/mol.

P4.3:
The triple bond in compound I is symmetric, and therefore is not IR-active, so there would be no absorbance in
the carbon-carbon triple bond range (2100-2250 cm-1). In compound II the presence of the fluorines makes the
triple bond asymmetric and IR-active, thus the alkyne peak will be observed. In compound III, we should see not
only the carbon-carbon triple bond peak but also an absorbance at approximately 3300 cm -1 due to stretching of
the terminal alkyne carbon-hydrogen bond.

P4.4:
All three spectra will have a strong carbonyl stretching peak, but the ester (compound C) carbonyl peak will be
observed at a shorter wavelength compared to the ketone (compound B) and the carboxylic acid (compound A).
In addition, Compound A will show a broad absorbance centered at approximately 3000 cm -1 due to carboxylic
acid O-H stretching, whereas in the spectrum of compound B we should see the broad absorbance centered at
approximately 3300 cm-1 from stretching of the alcohol O-H bond. Compound C will have no broad O-H stretching
absorbance.

P4.5:
All three compounds contain alkene functional groups. However, in compound Y the alkene is symmetric and
thus we would not expect to see an absorbance from C=C stretching in the 1620-1680 cm -1range.
We would expect to see this peak in the spectra of compounds X and Z; in addition we would expect to see, in
the compound X spectrum, a peak in the 3020 - 3080 cm -1 range due to stretching of the terminal alkene C-H
bonds.

P4.6:

P4.7:
The change in A340 is DA = 0.220. Using the expression = A/c, we can calculate that this represents a change in
the NADH concentration of 3.50 x 10-5 M. This is in a 1 mL solution, so 3.50 x 10 -8 mol have been used up over
the course of five minutes, or 7.00 x 10-9 mol (7.00 nmol) per minute on average.

P4.8:
Both starting compounds contain systems of conjugated pi bonds which absorb in the UV range. The
condensation reaction brings these two conjugated systems together to create a single, longer conjugated pi
system, which absorbs in the blue part of the visible spectrum.

P4.9:

P4.10:

The molecular ion and the fragments from all three of the typical ketone fragmentation patterns would all have
m/z = 98.

P4.11:
Both molecules contain alkene and ketone functional groups, however the degree of pi bond conjugation is
different. Therefore, UV would be the more useful technique to distinguish the two.

P4.12:
Both molecules are straight-chain alkanes with a single ketone group, so their IR spectra are expected to be very
similar and neither will absorb strongly in the UV range. However, the different positions of the ketone (at the
C4 vs C5 position) will result in the formation of fragments of different masses in an MS experiment.