12. structure determination: mass spectrometry and ... · pdf file12. structure determination:...

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NMR Spectroscopy

Introduction

Over the past fifty years nuclear magnetic resonance

spectroscopy, commonly referred to as nmr, has become the

most important technique for determining the structure of

organic compounds.

Although larger amounts of sample are needed than for mass

spectroscopy, nmr is non-destructive, and with modern

instruments good data may be obtained from samples weighing

less than a milligram.

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Principles of NMR Spectroscopy

NMR spectroscopy is basically another form of absorption

spectroscopy, similar to IR or UV spectroscopy.

Under appropriate conditions, a sample can absorb electromagnetic

radiation in the radio-frequency region at frequencies governed by the

characteristic of the sample.

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In NMR, absorption is a function of certain nuclei in

the molecule.

A plot of the frequencies of the absorption peaks

versus peak intensities constitutes an NMR

spectrum.

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All nuclei carry a charge.

In some nuclei this charge “spins” on the nuclear axis, and this

circulation of nuclear charge generates a magnetic dipole along

the axis.

The most important nuclei for organic structure determination

are 1H (ordinary hydrogen) and 13C, a stable nonradioactive

isotope of ordinary carbon.

Although 12C and 16O are present in most organic compounds,

they do not possess a spin and do not give NMR spectra.

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When nuclei with spin are placed between the poles of powerful

magnet, they align their magnetic fields with or against the field

of the magnet.

Nuclei aligned with the applied field have a slightly lower energy

than those aligned against the field.

By applying energy in the radio frequency range, it is possible to

excite nuclei in the lower energy spin state to the higher energy

spin state.

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The energy gap between the two spin states depends on the strength

of the applied magnetic field; the stronger the field, the larger the

energy gap.

Instruments currently in use have magnetic fields that range from about

1.4 to 14 tesla (T) (by comparison, the earth's magnetic field is only

about 0.0001 T).

At these field strengths, the energy gap corresponds to a radio

frequency of 60 to 600 MHz (megahertz; 1 MHz = 106 Hz or 106 cycles

per second).

This energy corresponds to 6-60 x 10-6 kcal/mole.

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Measuring an NMR Spectrum

A 1H-NMR spectrum is usually obtained by dissolving the

sample in some inert solvent that does not contain 1H nuclei.

Examples of such solvents are CCl4, or solvents with the

hydrogens replaced by deuterium, such as CDCl3, and

CD3COCD3.

A small amount of a reference compound is also added.

The solution, in a thin glass tube, is placed in the center of a

radio frequency (rf) coil, between the pole faces of a powerful

magnet.

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The nuclei align themselves with or against the field.

Continuously increasing amounts of energy is then applied to the

nuclei by the rf coil.

When this energy corresponds exactly to the energy gap between the

lower and higher energy spin states, it is absorbed by the nuclei.

At this point, the nuclei are said to be in resonance with the applied

frequency – hence the term nuclear magnetic resonance.

A plot of the energy absorbed by the sample against the applied

frequency of the rf coil gives an NMR spectrum.

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The Mechanism of Absorption

(a) A top precessing in the earth’s gravitational field; (b) the

precession of a spinning nucleus resulting from the influence of

an applied magnetic field.

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The Mechanism of Absorption

The nuclear magnetic resonance process: absorption occurs

when ν = ω.

Where ν is applied radio frequency and ω is the angular

frequency of precession.

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Population densities of nuclear spin

states

For a proton, if the applied magnetic field

strength is 1.41 Tesla, resonance occurs at

about 60 MHz.

The energy different between the two spin

states is about 6 x 10-6 kcal/mole.

At room temperature both the states are

almost equally populated.

There is slight excess of nucleii in the lower

energy state.

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Population densities of nuclear spin

states

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In practice, there are two ways by which the resonance

frequencies of 1H nuclei can be determined.

Because the magnetic field strength and the size of the energy

gap between nuclear spin states are directly related, either the

magnetic field strength or the rf can be varied.

In earlier NMR spectrometers a constant radio frequency was

applied, the strength of the applied magnetic field was varied,

and different nuclei resonated at different magnetic field

strengths.

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In modern Fourier transform (FT-NMR) spectrometer, the

applied magnetic field is held constant, and the radio frequency

is varied.

The instrument computer uses a mathematical process called

Fourier transformation to sort the signal that is produced into the

resonance rfs of the different 1H nuclei.

Whether it is the magnetic field field strength or the rf that is

varied, this variable increases from left to right in the recorded

spectra.

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The NMR instrument

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Chemical Shifts and Peak Areas

Not all 1H nuclei flip their spins at precisely the same radio frequency because they may differ in chemical (and, more particularly, electronic) environment.

The NMR spectrum of

p-xylene:

The spectrum is very

simple and consists

of two peaks.

The positions of the

peaks are measured

in (delta) units from the peak of a reference compound, which is

tetramethylsilane (TMS), (CH3)4Si.

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The reasons for selecting TMS as a

reference compound

All 12 nuclei of its hydrogens are equivalent, so it

shows only one sharp NMR signal, which serves as a

reference point.

Its hydrogen signals appear at higher field than do

most 1H signals in other organic compounds, thus

making it easy to identify the TMS peak.

TMS is inert, so it does not react with most organic

compounds, and it is low boiling and can be removed

easily at the end of a measurement.

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Most organic compounds have peaks downfield (at low field) from TMS and are given positive values.

low field high field

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A value of 1.00 means that a peak appears 1 part per million (ppm) downfield from the TMS peak.


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If the spectrum is measured at 60 MHz (60 x 106 Hz), then 1 ppm is 60 Hz (one-millionth of 60 MHz) downfield from TMS.

If the spectrum is run at 100 MHz, a value of 1 ppm is 100 Hz downfield from TMS, and so on.


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The chemical shift of a particular kind of 1H signal is its value with respect to TMS.

It is called a chemical shift because it depends on the chemical environment of the hydrogens.

The chemical shift is independent of the instrument on which it is measured.

Chemical shift = = distance of peak from TMS, in Hz

-------------------------------------------- ppm

spectrometer frequency in MHz

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In the spectrum of p-xylene, we see a peak at 2.30 and another at 7.10.

It seems reasonable that these peaks are caused by two different “kinds” of 1H nuclei in the molecule: the methyl hydrogens and the aromatic ring hydrogens.

How can we tell which is which?


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One way to identify the hydrogens is to integrate the area under each peak.

The peak area is directly proportional to the number of 1H nuclei responsible for the particular peak.

All the commercial NMR spectrometers are equipped with electronic integrators that can print an integration line over the peaks.


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The ratio of heights of the vertical parts of this line is the ratio of peak areas..

The areas of the peaks at 2.30 and 7.10 in p-xylene spectrum give a ratio of 3 : 2 (or 6 : 4).

These areas allow us to assign the peak at 2.30 to the methyl hydrogens and the peak at 7.10 to the four aromatic ring hydrogens.


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A more general way to assign peaks is to compare chemical shifts with those of similar protons in a known reference compound.

For example, benzene has six equivalent hydrogens and shows a single peak in its 1H NMR spectrum at 7.24.

Other aromatic compounds also show a peak in this region. We can conclude that most aromatic ring hydrogens will have chemical shifts at about 7.

Similarly, most CH3 — Ar hydrogens appear at 2.2 – 2.5.

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Chemical Shift

6 regions of NMR spectrum

The chemical shifts of 1H nuclei in various chemical environments

have been determined by 1H NMR spectra of a large number of

compounds with known, relatively simple structures.

The chart above gives the chemical shifts for several common

types of 1H nuclei.

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Table of chemical shifts

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Factors that influence chemical shifts

The electronegativity of groups in the immediate environment of the 1H nuclei.

Electron withdrawing groups generally cause a downfield chemical shift.

Compare, for example, the following chemical shifts:

CH3 CH2Cl CHCl2

~ 0.9 ~ 3.7 ~ 5.8

Electrons in motion near a 1H nucleus create a small magnetic field in its microenvironment that tends to shield the nucleus from the externally applied magnetic field.

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Electrons in motion near a 1H nucleus create a small magnetic field in its microenvironment that tends to shield the nucleus from the externally applied magnetic field.

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CH3 CH2Cl CHCl2

~ 0.9 ~ 3.7 ~ 5.8

Chlorine is an electron-withdrawing group.

Withdrawal of electron density by the chlorine therefore “deshields” the nucleus, allowing it to flip its spin at a lower applied external field or lower frequency.

The more chlorines, the larger the effect.

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The presence of electrons

Hydrogens attached to a carbon that is part of a multiple bond or aromatic ring usually appears downfield from hydrogens attached to saturated carbons.

The pi-electrons associated with

a benzene ring provide a striking

example of this phenomenon.

The electron cloud above and below the plane of the ring circulates in reaction to the external field so as to generate an opposing field at the

center of the ring and a supporting field at the edge of the ring.

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This kind of spatial variation is called anisotropy, and it is common to nonspherical distributions of electrons.

Regions in which the induced

field supports or adds to the

external field are said to be

deshielded, because a slightly

weaker external field will bring

about resonance for nuclei in

such areas.

However, regions in which the induced field opposes the external field are termed shielded because an increase in the applied field is needed for resonance.

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Examples of Anisotropy Influences on

Chemical Shift

The structural constraints of the bridging chain require the middle two methylene groups to lie over the face of the benzene ring, which is a nmr shielding region.

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Spin – Spin Splitting

Many compounds give spectra that show more complex peaks than just single peaks (singlets) for each type of hydrogen.

Based on chemical shifts, the 1H NMR spectrum of diethyl ether is expected to have two lines:

one in the region of 0.9 for the six equivalent CH3 Hydrogens and one at about 3.5 for the four equivalentCH2 hydrogens adjacent to the oxygen atom, with relative areas 6 : 4.

In the spectrum, we see absorptions in each of these regions, with the expected total area ratio.

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But, we do not see singlets!

Instead, the methyl signal is split into three peaks, a triplet, with relative areas 1:2:1; and the methylene signal is split into four peaks, a quartet, with relative areas 1:3:3:1.

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Spin-spin splittings, tell us quite a bit about molecular structure.

Each 1H nucleus in the molecule acts as a tiny magnet, and each

hydrogen “feels” not only the very large applied magnetic field but also

a tiny field due to its neighboring hydrogens.

When 1H nuclei on one carbon is excited, the 1H nuclei on neighboring

carbons can be in either the lower or the higher spin state, with nearly

equal probabilities.

Due to this, the magnetic field of the nuclei whose peak is observed is

perturbed slightly by the tiny fields of its neighboring 1H nuclei.

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The splitting pattern can be predicted by the n + 1 rule: if a 1H nucleus

or a set of equivalent 1H nuclei has n 1H neighbors with a substantially

different chemical shift, its NMR signal will be split into n + 1 peaks.

In diethyl ether, each CH3 hydrogen has two 1H neighbors (on the CH2

group).

Therefore, the CH3 signal is split into 2 + 1 = 3 peaks.

At the same time, each CH2 hydrogen has three 1H neighbors (on the

CH3 group). The CH2 signal is therefore split into 3 + 1 = 4 peaks.

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n + 1 rule for signal splitting

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In the spectrum of 1,1-dichloroethane shown below, it is clear that

the three methyl hydrogens (red) are coupled with the single

methyne hydrogen (orange) in a manner that causes the former to

appear as a doublet and the latter as a quartet.


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The statistical distribution of spins within each set explains both the n+1

rule and the relative intensities of the lines within a splitting pattern.

The action of a single neighbouring proton is easily deduced from the fact

that it must have one of two possible spins. Interaction of these two spin

states with the nuclei under observation leads to a doublet located at the

expected chemical shift.


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The corresponding action of the three protons of the methyl group requires a

more detailed analysis.

In the display of this interaction four possible arrays of their spins are shown.

The mixed spin states are three times as possible as the all +1/2 or all _1/2

collection.

Consequently, we expect four signals, two above the chemical shift and two

below it. This spin analysis also suggests that the intensity ratio of these

signals will be 1:3:3:1.


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A simple way of estimating the relative intensities of the lines in a first-order

coupling pattern is shown below.

This array of numbers is known as Pascal's triangle, and is easily extended

to predict higher multiplicities.


Pascal’s Triangle

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Coupling Constant

1H nuclei that split one another’s signals are said to be coupled.

The extent of the coupling, or the number of hertz by which the signals split, is

called the coupling constant (abbreviated J).


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Coupling Constant

Spin – spin splitting falls off with distance.

Whereas hydrogens on adjacent carbons may show appreciable

splitting ( J = 6 – 8 Hz), hydrogens farther apart hardly “feel” each

other’s presence ( J = 0 – 1 Hz).

Coupling constants can even be used at times to distinguish between

cis – trans isomers or between positions of substituents on a benzene

ring.

Chemically equal 1H nuclei do not split each other. For example,

BrCH2CH2Br shows only a sharp singlet in its 1H NMR spectrum for

all four hydrogens.

Measuring Coupling Constant

Each ppm of chemical shift represents 60Hz.

There are 12 grid lines per ppm, each grid line represents 60Hz/12 = 5

Hz.

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Measuring coupling constant

The spacing between the component peaks is approximately 1.5

chart divisions.

Therefore, J = 1.5 div x 5 Hz/1 div = 7.5 Hz

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Measuring coupling constant

Most aliphatic protons in acylic systems, the magnitutes of

coupling constants are always near 7.5 Hz.

The coupling constants of the groups of protons that split one

another are identical within experimental error.

This information is useful in interpreting a spectrum that may

have several multiplets, each with a different coupling constant.

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Measuring coupling constants on

modern FT-NMR spectrometers

Coupling constants are determined by printing Hertz

values directly on the peaks.

The values are subtracted to determine the coupling

constants.

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Multiplet Skewing (leaning)

Multiplet skewing can sometimes be used to link interacting

multiplets.

There is a tendency for the outermost lines of a multiplet to have

nonequivalent heights.

If arrows are drawn on both multiplets in the directions of their

respective skewing, these arrows will point at each other.

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NMR spectra of alcohols

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The chemical shift of –OH hydrogen is variable.

Its position depending on concentration, solvent,

temperature, and presence of water, or acidic or

basic impurities.

This peak can be found anywhere in the range of 0.5-

5.0 ppm.

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The variability of this absorption is dependent on the

rates of –OH proton exchange and the amount of

hydrogen bonding in the solution.

The –OH hydrogen is usually not split by hydrogens

on the adjacent carbon (-CH-OH) because rapid

exchange decouples this interaction.

Deuterium exchange for identifying

the –OH absorption

A drop of D2O is added to the NMR tube containing

the alcohol solution.

After shaking the sample and sitting for few minutes,

the NMR spectrum is recorded.

The disappearance of the –OH signal in the spectrum

is used to confirm the presence of –OH group in the

sample.

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Deuterium exchange for identifying

amines and carboxylic acids

Amino hydrogens exhibit deuterium

exchange.

Carboxylic hydrogen exhibit deuterium

exchange.

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Interpreting NMR spectra

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