Basic Interpretation of a 1H NMR Spectrum

 Formula of the compound – identify whether saturated (2n+2 of no. of H atoms where n = no. of C atoms) or unsaturated

 Number of signals – indicate how many ‘chemically non-equivalent’ protons are present

 Position of signals – indicates chemical shift, magnetic (electronic) environment of protons

 Relative intensity of signals – Proportional to number of protons present

 Splitting of signals (spin-spin coupling) – indicates the number of nearby nuclei (usually protons)

Example 1

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 Prediction of the structure of Example 1

 Formula of the compound – C5H12O2 – saturated compound

 Number of signals – Two signals – means two different types of protons

 Position of signals – ~1.2 and ~3.2 ppm – indicates two different types of aliphatic C-H protons

 Relative intensity of signals – 1:1 – indicates 12 protons are divided into two sets of ‘chemically non-equivalent’ six protons

 Splitting of signals (spin-spin coupling) – two singlets – indicates no nearby coupling nuclei, i.e., two sets of non-
equivalent’ –CH3 protons

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 Prediction of the structure of Examples 2 and 3

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 Prediction of the structure of Examples 4 and 5

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 Prediction of the structure of Examples 6 and 7

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 Freezing of H in -OH groups
Under routine conditions, rapid intermolecular exchange of the OH protons of alcohols often prevents their coupling with adjacent
hydrogens from being observed. Intermediate rates of proton exchange lead to a broadening of the OH and coupled hydrogen
signals, a characteristic that is useful in identifying these functions

The 60 MHz proton nmr spectrum of pure (neat) methanol exhibits two signals, as expected. At 30° C these signals are sharp
singlets located at δ 3.35 and 4.80 ppm, the higher-field methyl signal (magenta) being three times as strong as the OH signal
(orange) at lower field. When cooled to -45 °C, the larger higher-field signal changes to a doublet (J = 5.2 Hz) having the same
chemical shift. The smaller signal moves downfield to δ 5.5 ppm and splits into a quartet (J = 5.2 Hz). The relative intensities of
the two groups of signals remains unchanged.

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 Spin Decoupling in 1H NMR Spectroscopy
We have noted that rapidly exchanging hydroxyl hydrogens are not spin-coupled to adjacent C-H groups. As each exchange
occurs, there will be an equal chance of the new proton having a +1/2 or a _1/2 spin (remember that the overall populations of
the two spin states are nearly identical). Over time, therefore, the hydroxyl hydrogen behaves as though it is rapidly changing its
spin, the adjacent nuclei see only a zero spin average from it. If we could cause other protons in a molecule to undergo a similar
spin averaging, their spin-coupling influence on adjacent nuclei would cease. Such NMR experiments are possible, and are
called spin decoupling.

When a given set of nuclei is irradiated with strong rf energy at its characteristic Larmor frequency, spin saturation and rapid
interconversion of the spin states occurs. Neighbouring nuclei with different Larmor frequencies are no longer influenced by
specific long-lived spins, so spin-spin signal splitting of the neighbours vanishes.

The following spectrum of 1-nitropropane may be used to illustrate the spin decoupling technique. The three distinct sets of
protons in this molecule generate three resonance signals (two triplets and a broad sextet).

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 The Influence of Magnetic Field Strength
It is important to remember that structurally different sets of nuclei do not always produce distinctly different signals in an NMR spectrum. For example,
the hydrocarbon octane has four different sets of protons, as shown in the following formula: CH3CH2CH2CH2CH2CH2CH2CH3. Now methyl hydrogens
have a smaller chemical shift than methylene hydrogens, so methyl groups (colored black here) can usually be distinguished. However, the chemical
shifts of the different methylene groups (blue, red & green) are so similar that many NMR spectrometers will not resolve them. Consequently, a 90 MHz
proton spectrum of octane shows a distorted triplet at δ 0.9 ppm, produced by the six methyl protons, and a strong broad singlet at δ 1.2 ppm coming
from all twelve methylene protons.
A similar failure to resolve structurally different hydrogen atoms occurs in the case of alkyl substituted benzene rings. The chemical shift difference
between ortho, meta and para hydrogens in such compounds is often so small that they are seen as a single resonance signal in an NMR spectrum.
The 90 MHz spectrum of benzyl alcohol in chloroform-d solution provides an instructive example, shown below. A broad strong signal at δ 7.24 ppm is
characteristic of the aromatic protons on alkylbenzenes. Since the chemical shifts of these hydrogens are nearly identical, no spin coupling is
observed.
If the magnetic field strength is increased to 400 Mz (lower spectrum) the aromatic protons are more dispersed (orange, magenta and green signals),
and the spin coupling of adjacent hydrogens (J = 7.6 Hz) causes overlap of the signals (gray shaded enlargement).

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 An Example of Extensive Spin-Splitting

Here it is clear that each of the five hydrogen atoms in the molecule is structurally unique, and is producing a separate signal.

This spectrum has several interesting features. First, hydrogens A, B & C are clearly
different, and are spin-coupled to each other. Hydrogens B & C are geminally related,
whereas A is oriented to B & C in a vicinal manner. Since JAB and JBC are similar, the
HB signal is a broad triplet. Although hydrogens D & E might seem identical at first
glance, they are diastereotopic, and should therefore have different chemical shifts.
The DE geminal coupling constant is 11.7 Hz, and D & E also couples with A; so each
of these hydrogens appears as a doublet of doublets.

The splitting of the HA signal is complex and not immediately obvious. The diagram on
the right shows the consequences of the four operating coupling constants.

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  13C NMR Spectroscopy – why it is needed?
The power and usefulness of 1H NMR spectroscopy as a tool for structural analysis should be evident from the past discussion.
Unfortunately, when significant portions of a molecule lack C-H bonds, no information is forthcoming. Examples include
polychlorinated compounds such as chlordane, polycarbonyl compounds such as croconic acid, and compounds incorporating
triple bonds (structures below, orange colored carbons).

Even when numerous C-H groups are present, an unambiguous interpretation of a proton NMR spectrum may not be possible.
The following diagram depicts three pairs of isomers (A & B) which display similar proton NMR spectra. Although a careful
determination of chemical shifts should permit the first pair of compounds (blue box) to be distinguished, the second and third
cases (red & green boxes) might be difficult to identify by proton NMR alone.

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  13C NMR Spectroscopy – why it is needed?
These difficulties would be largely resolved if the carbon atoms of a molecule could be probed by NMR in the same fashion as
the hydrogen atoms. Since the major isotope of carbon (12C) has no spin, this option seems unrealistic. Fortunately, 1.1% of
elemental carbon is the 13C isotope, which has a spin I = 1/2, so in principle it should be possible to conduct a 13carbon NMR
experiment. It is worth noting here, that if much higher abundances of 13C were naturally present in all carbon compounds,
proton NMR would become much more complicated due to large one-bond coupling of 13C and 1H.

Many obstacles needed to be overcome before carbon NMR emerged as a routine tool:

i) As noted, the abundance of 13C in a sample is very low (1.1%), so higher sample concentrations are needed.
ii) The 13C nucleus is over fifty times less sensitive than a proton in the NMR experiment, adding to the previous difficulty.
iii) Hydrogen atoms bonded to a 13C atom split its NMR signal by 130 to 270 Hz, further complicating the NMR spectrum.

The most important operational technique that has led to successful and routine 13C NMR spectroscopy is the use of high-field
pulse technology coupled with broad-band heteronuclear decoupling of all protons. The results of repeated pulse sequences are
accumulated to provide improved signal strength. Also, for reasons that go beyond the present treatment, the decoupling
irradiation enhances the sensitivity of carbon nuclei bonded to hydrogen.

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 Pulsed Fourier Transform (FT) Spectroscopy
In a given strong external magnetic field, each structurally distinct set of hydrogens in a molecule
has a characteristic resonance frequency, just as each tubular chime in percussion instrument
has a characteristic frequency. The drawing on the right depicts a set of four chimes, with the
frequency of each designated by a colored sine wave. To discover the frequency of a chime we
can strike it with a mallet and measure the sound emitted. This procedure can be repeated for
each chime in the group so that all the characteristic frequencies are identified.

An alternative means of acquiring the same information is to strike all the chimes simultaneously, and to subject the complex
collection of frequencies produced to mathematical analysis. In the following diagram the four frequencies assigned to our set of
chimes are added together to give a complex summation wave. This is a straightforward conversion; and the reverse
transformation is readily accomplished, provided the combination signal is adequately examined and characterized.

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 Principles of Pulsed FT NMR
A CW NMR spectrometer functions by irradiating each set of distinct nuclei in turn, a process analogous to striking each chime
independently. For a high resolution spectrum this must be done slowly, and a 12 ppm sweep of the proton region takes from 5 to
10 minutes. It has proven much more efficient to excite all the proton nuclei in a molecule at the same time, followed by
mathematical analysis of the complex rf resonance frequencies emitted as they relax back to the equilibrium state. This is the
principle on which a pulse Fourier transform spectrometer operates. By exposing the sample to a very short (10 to 100 μsec),
relatively strong (about 10,000 times that used for a CW spectrometer) burst of rf energy along the x-axis, all of the protons in the
sample are excited simultaneously.

The overlapping resonance signals generated as the excited protons relax are
collected by a computer and subjected to a Fourier transform mathematical
analysis. As shown in the diagram on the left, the Fourier transform analysis,
abbreviated FT, converts the complex time domain signal emitted by the sample
into the frequency (or field) domain spectrum we are accustomed to seeing. In this
fashion a complete spectrum can be acquired in a few seconds.

Because the relaxation mechanism is a first order

process, the rf signal emitted by the sample
decays exponentially. This is called a free
induction decay signal, abbreviated FID.
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 Example of more informative 13C NMR than a 1H NMR spectrum
Since, the FID signal collected after one pulse, may be stored and averaged with the FID's from many other identical pulses prior to the Fourier
transform, the NMR signal strength from a small sample may be enhanced to provide a useable spectrum. This has been essential to acquiring
spectra from low abundance isotopes, such as 13C. In practice, the pulse FT experiment has proven so versatile that many variations of the
technique, suited to special purposes, have been devised and used effectively.

When acquired by FT method, the carbon NMR spectrum of a compound displays a single sharp signal for each structurally distinct carbon atom in
a molecule (remember, the proton couplings have been removed). The spectrum of camphor, shown below, is typical. Furthermore, a comparison
with the 1H NMR spectrum on the right illustrates some of the advantageous characteristics of carbon NMR. The dispersion of 13C chemical shifts is
nearly twenty times greater than that for protons, and this together with the lack of signal splitting makes it more likely that every structurally distinct
carbon atom will produce a separate signal. The only clearly identifiable signals in the proton spectrum are those from the methyl groups. The
remaining protons have resonance signals between 1.0 and 2.8 ppm from TMS, and they overlap badly due to spin-spin splitting.

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 Characteristic Features of 13C NMR Spectroscopy

 The chemical shift of 13C NMR is wider (0-230 ppm relative to TMS) compared to that of 1H NMR (0-20 ppm relative to TMS)

 13C-13C coupling is negligible because of low natural abundance (1.1%) of 13C in the compound.

 The gyromagnetic ratio of a 13C nucleus (1.404) is much less than that of a 1H nucleus (5.585), thus making it much less
sensitive than a 1H nucleus.

 In proton decoupled 13C NMR, each magnetically non-equivalent carbon gives a single sharp peaks that does undergo further
spitting in presence of 19F or 31P nuclei.

 The area under the peak in 13C NMR is not necessarily proportional to the number of carbon responsible for the signal – thus
integrations may not be accurate.

 Spectra can be made more quantitative if necessary by allowing sufficient time for the nuclei to relax between repeat scans.

 In proton coupled 13C NMR spectrum the signal of each carbon or group of magnetically equivalent carbon is split by the
proton bonded directly to that carbon and n+1 rule is followed.

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Pavia, Lampman, Kriz, Vyvyan; Spectroscopy, Cengage learning, India Ed., 2007
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  13C Chemical Shift Ranges?

* For samples in CDCl3 solution. The δ scale is relative to TMS at δ=0.

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 Examples where 13C NMR is superior to 1H NMR

The isomeric pairs previously cited as giving very similar proton nmr spectra are now seen to be distinguished by carbon nmr. In the
example on the left below (blue box), cyclohexane and 2,3-dimethyl-2-butene both give a single sharp resonance signal in the proton nmr
spectrum (the former at δ 1.43 ppm and the latter at 1.64 ppm). However, in its carbon nmr spectrum cyclohexane displays a single signal
at δ 27.1 ppm, generated by the equivalent ring carbon atoms (colored blue); whereas the isomeric alkene shows two signals, one at δ
20.4 ppm from the methyl carbons (colored brown), and the other at 123.5 ppm (typical of the green colored sp2 hybrid carbon atoms).

The C8H10 isomers in the center (red) box have pairs of homotopic carbons and hydrogens, so symmetry should simplify their nmr
spectra. The fulvene (isomer A) has five structurally different groups of carbon atoms (colored brown, magenta, orange, blue and green
respectively) and should display five 13C nmr signals (one near 20 ppm and the other four greater than 100 ppm). Although ortho-xylene
(isomer B) will have a proton nmr very similar to isomer A, it should only display four 13C nmr signals, originating from the four different
groups of carbon atoms (colored brown, blue, orange and green). The methyl carbon signal will appear at high field (near 20 ppm), and
the aromatic ring carbons will all give signals having δ > 100 ppm. Finally, the last isomeric pair, quinones A & B in the green box, are
easily distinguished by carbon nmr. Isomer A displays only four carbon nmr signals (δ 15.4, 133.4, 145.8 & 187.9 ppm); whereas, isomer
B displays five signals (δ 15.9, 133.3, 145.8, 187.5 & 188.1 ppm), the additional signal coming from the non-identity of the two carbonyl
carbon atoms (one colored orange and the other magenta).
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 Types of Decoupling in 13C NMR Spectroscopy
The most common modes of recording 13C spectra are proton-noise or broadband decoupling, off-resonance decoupling, and
gated decoupling. These modes are meant to address the large J values for 13C - H (110–320 Hz), 13C - C - H (5–60 Hz), and 13C
- C - C - H (5–25 Hz) which otherwise make completely proton coupled 13C spectra difficult to interpret.

With proton-noise decoupling, in which most spectra are run, a noise decoupler strongly irradiates the sample with a broad
(approximately 1000 Hz) range of radio frequencies covering the range (such as 100 MHz for a 23,486 gauss field) at which
protons change their nuclear spin. The rapid changes in proton spin create an effective heteronuclear decoupling, increasing
carbon signal strength on account of the Nuclear Overhauser Effect (NOE) and simplifying the spectrum so that each non-
equivalent carbon produces a singlet peak. The relative intensities are unreliable because some carbons have a larger spin-
lattice relaxation time and others have weaker NOE enhancement.

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L. D. S. Yadav (2013), Organic Spectroscopy, Springer. pp. 197–1990 www.iitjammu.ac.in
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 Types of Decoupling in 13C NMR Spectroscopy
With off-resonance decoupling, the noise decoupler irradiates the sample at 1000–2000 Hz upfield or 2000–3000 Hz downfield of
the proton resonance frequency. This retains couplings between protons immediately adjacent to 13C atoms but most often
removes the others, allowing narrow multiplets to be visualized with one extra peak per bound proton (unless bound methylene
protons are non-equivalent, in which case a pair of doublets may be observed).

In gated decoupling, the noise decoupler is gated on early in the free induction delay but gated off for the pulse delay. This largely
prevents NOE enhancement, allowing the strength of individual 13C peaks to be meaningfully compared by integration, at a cost of
half to two-thirds of the overall sensitivity.

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Distortionless enhancement by polarization transfer (DEPT) NMR
DEPT NMR is an NMR method used for determining the presence of primary, secondary and tertiary carbon atoms. It is a gated
decoupling NMR technique. The DEPT experiment differentiates between CH, CH2 and CH3 groups by variation of the selection
angle parameter (the tip angle of the final 1H pulse): 135° angle gives all CH and CH3 in a phase opposite to CH2; 90° angle
gives only CH groups, the others being suppressed; 45° angle gives all carbons with attached protons (regardless of number) in
phase (similar to a 1H decoupled 13C NMR).

Signals from quaternary carbons and other carbons with no attached protons are always absent.

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Doddrell, D.M.; Pegg, D.T.; Bendall, M.R. (1982). "Distortionless enhancement of NMR signals by polarization transfer". J. Magn. Reson. 48 (2): 323–327
Example of final structure determination by DEPT NMR
The 1H and 13C NMR spectra of a compound with formula C5H9Br is shown below. The DEPT experimental results are also provided in the table

First, determine the hydrogen deficiency index. Replacing the Br in C5H9Br with a H we get C5H10 which corresponds to one degree of
unsaturation. Therefore, the compound has a double bond or a ring.

Now, looking at signal at about 4.7 ppm in the proton, and the ones above 100 on the carbon, we know that it must be a double bond rather than a
ring.

Next, look at the signal splitting in 1H NMR; two triplets indicate a -CH2-CH2– fragment which is connected to Br on one end since it is downfield
(3.3 ppm). And the other CH2 must be connected to the double bond since the signal is still more downfield than if it was a regular alkyl group.
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Keeler, James (2010). Understanding NMR Spectroscopy (2nd ed.). John Wiley & Sons. p. 457. ISBN 978-0-470-74608-0.
 Example of final structure determination by DEPT NMR

So, let’s put down the groups we have so far:

Two of these X groups must be hydrogens because of the integration of the signal at ~4.7 ppm.

The other X group is a methyl group which we can deduce from the integration.

And now the interesting part related to DEPT. There are three combinations of putting two hydrogens and a methyl group on
the double bond:

All of these would be good candidates based on the data from the proton and carbon NMR. But only the last structure matches
the data from the DEPT experiments which indicate the presence of three CH2 groups (three negative signals in DEPT-135)
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 Example of final structure determination by DEPT NMR

The structure of a double bond can be analyzed

using the J coupling values and a powerful NMR
spectrometer will give a resolution good enough to
exclude the other candidates base on the coupling.
However, DEPT makes things easier without the
need for a lot of complicated analysis.

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Nuclear Overhauser Effect (NOE)
Change (positive or negative) of resonance line intensity due to dipolar cross relaxation from neighbouring spins with perturbed
energy level populations.

Consider a molecule in which two protons are close enough (< 5 Å) to allow through space interactions of the
fluctuating magnetic vector; for this effect the number of intervening bonds between the two concerned
protons have no significance. In this molecule if we irradiate Hb then this proton gets stimulated and this
stimulation is transferred through space to the relaxation mechanism of Ha

To understand the nature of NOE, we’ve to look at two spin systems I1 and I2.

The possible transitions for these two spin systems can be classified into three groups

(1) W1 transitions involving a spin flip of only one of the two spins (either I1 or I2) corresponding to relaxation of spin
(2) W0 transition involving a simultaneous spin flip α→β for one spin and β→α for the other one (i.e., in summation a zero-
quantum transition)
(3) W2 transition involving a simultaneous spin flip of both spins in the same direction, corresponding to a net double-quantum
transition.

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Noggle, Joseph H.; Schirmer, Roger E. (1971). The Nuclear Overhauser Effect: Chemical Applications. Academic Press
 Nuclear Overhauser Effect (NOE)
With the I1 polarization going back from saturation to the Boltzmann equilibrium, the W0 mechanism will cause the neighbouring
(so far unperturbed) spin to deviate from its Boltzmann equilibrium towards a decrease in α, β population difference. After a 90o
pulse this will result in a decrease in signal intensity for I2 – a ‘negative NOE effect’.

On the other hand the W2 mechanism, will cause the population difference of the undisturbed spin I2 to increase, corresponding
to an increase in signal intensity – a ‘positive NOE effect’.

In the example on right, the proton designated as {H} shows two different
sets of NOEs depending on the isomerization state (cis or trans) of the
switchable azo groups. In the trans state proton {H} is far from the phenyl
group showing blue coloured NOEs; while the cis state holds proton {H} in the
vicinity of the phenyl group resulting in the emergence of new NOEs (show in
red).

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 Mass Spectrometry

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of ions. The results are typically
presented as a mass spectrum, a plot of intensity of flying ion signal as a function of the mass-to-charge ratio. Mass
spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures.
These spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules,
and to elucidate the chemical identity or structure of molecules and other chemical compounds.

In a typical MS procedure, a sample, which may be solid, liquid, or gaseous,

is ionized, for example, by bombarding it with electrons. This may cause

Intensity (A.U.)
some of the sample's molecules to break into charged fragments or simply
become charged without fragmenting. These ions are then separated
according to their mass-to-charge ratio, for example, by accelerating them
and subjecting them to an electric or magnetic field: ions of the same mass-
to-charge ratio will undergo the same amount of deflection.[1] The ions are
detected by a mechanism capable of detecting charged particles, such as an
electron multiplier. Results are displayed as spectra of the signal intensity of
detected ions as a function of the mass-to-charge ratio. The atoms or
molecules in the sample can be identified by correlating known masses (e.g. m/z
an entire molecule) to the identified masses or through a characteristic
fragmentation pattern.

[1] Sparkman, O. David (2000). Mass spectrometry desk reference. Pittsburgh: Global View Pub. ISBN 978-0-9660813-2-9
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Instrumentation of MS

The three essential functions of a mass spectrometer, and the

associated components, are:

1. A small sample is ionized, usually to cations by loss of an

electron. The Ion Source
2. The ions are sorted and separated according to their mass and
charge. The Mass Analyzer
3. The separated ions are then measured, and the results
displayed on a chart. The Detector
Because ions are very reactive and short-lived, their formation and manipulation must be conducted in a vacuum. Atmospheric
pressure is around 760 torr (mm of mercury). The pressure under which ions may be handled is roughly 10-5 to 10-8 torr (less than
a billionth of an atmosphere). Each of the three tasks listed above may be accomplished in different ways. In one common
procedure, ionization is effected by a high energy beam of electrons, and ion separation is achieved by accelerating and focusing
the ions in a beam, which is then bent by an external magnetic field. The ions are then detected electronically and the resulting
information is stored and analyzed in a computer. The heart of the spectrometer is the ion source. Here molecules of the sample
(black dots) are bombarded by electrons (light blue lines) issuing from a heated filament. This is called an EI (electron-impact)
source. Gases and volatile liquid samples are allowed to leak into the ion source from a reservoir (as shown). Non-volatile solids
and liquids may be introduced directly. Cations formed by the electron bombardment (red dots) are pushed away by a charged
repeller plate (anions are attracted to it), and accelerated toward other electrodes, having slits through which the ions pass as a
beam. Some of these ions fragment into smaller cations and neutral fragments. A perpendicular magnetic field deflects the ion
beam in an arc whose radius is proportional to the mass of each ion. Lighter ions are deflected more than heavier ions. By varying
the strength of the magnetic field, ions of different mass can be focused progressively on a detector fixed at the end of a curved
tube (also under a high vacuum).
Instrumentation of MS
When a high energy electron collides with a molecule it often ionizes it by knocking away one of the molecular electrons (either
bonding or non-bonding). This leaves behind a molecular ion (colored red in the following diagram). Residual energy from the
collision may cause the molecular ion to fragment into neutral pieces (colored green) and smaller fragment ions (colored pink
and orange). The molecular ion is a radical cation, but the fragment ions may either be radical cations (pink) or carbocations
(orange), depending on the nature of the neutral fragment.

Mass spectrometry is an analytical technique that involves the study in the gas phase of ionized molecules with the aim of one or
more of the following:

•Molecular weight determination

•Structural characterization
•Gas phase reactivity study
•Qualitative and quantitative analysis of components in a mixture.

Atomic and molecular masses are assigned relative to the mass of the carbon isotope, 12C, whose atomic weight is defined as
exactly 12. The actual mass of 12C is 12 daltons, with one dalton is equal to 1.661 x10-24 g. The mass of a molecule or an ion can
be presented in daltons (Da) or kilodaltons (kDa).