Structure

Determination II
NMR spectroscopy
Topics
● Theory and instrumentation of Nuclear Magnetic Resonance spectroscopy
● Determining whether nuclei are chemically distinct; diastereotopic protons
● Chemical shifts in NMR spectra; table of 1H chemical shifts
● Ring Currents and their impact on 1H chemical shifts
● Signal integration in 1H NMR
● Spin-spin splitting in 1H NMR: common and complex splitting patterns
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● C NMR Spectroscopy: FTNMR, interpreting spectra
● Advanced NMR techniques: DEPT and 2D NMR methods
● Biochemical and medical applications
Chapter Objectives
● Develop a basic understanding of nuclear magnetic resonance spectroscopy
● Understand what kind of information can be obtained from 1H and 13C NMR
spectra
● Recognize the regions of NMR spectra where specific types of proton and carbons
atoms will appear: alkane, alkene, alkyne, aromatic, carbonyl
● Learn readily recognizable splitting patterns in 1H NMR and apply them to
structural features
● Use NMR spectroscopy to identify molecular structure, both by itself and in
conjunction with other spectroscopic methods
What does NMR do?
● NMR spectroscopy provides structural information on molecules
● While IR identifies functional groups, NMR provides information on the skeleton of
the molecule: how the atoms are connected to each other.
● NMR can be used to examine several different types of atom: 1H,13C, 19F, 14N, 31P,
etc.
● A large variety of different types of NMR experiments have been developed to view
different aspects of molecular structure.
● NMR has direct applications to medicine, where it is more commonly encountered
as MRI.
NMR Principles
● Atomic nuclei spin in their molecules; several atoms generate a net magnetic
moment while spinning; think of these as atom sized bar magnets.
● In a normal environment, these magnets have their N and S poles randomly
distributed; however, if a large external magnetic field is applied, most of these
magnets will be aligned with and opposed to the external field.
NMR Principles (contd)
● The aligned nuclei are in the lower energy α (alpha) state, while the opposed nuclei
are in the higher energy β (beta) state.
● Radiation in the radio wave region of the EM spectrum will excite nuclei from α to
β; these nuclei will then decay back; this excitation/decay cycle is called resonance.
NMR Instrumentation
Based on the principle of nuclear magnetic resonance, an NMR spectrometer consists of:

1. A powerful magnet, the field of which the sample is placed in

2. An adjustable radio wave source to excite the sample
3. A detector that can receive the radio waves from the resonance decay

The key to NMR spectroscopy is that the radio wave frequency of a nucleus is dependent
on its environment, and the intensity of the signal is dependent on the number of nucleii
in the same environment.
NMR Signal Frequencies
● The radio frequency required for resonance for a given nucleus is dependent on
the strength of the external magnetic field; stronger fields lead to a greater ΔE
between α and β states.
● Stronger magnetic fields give increased resolution for NMR spectra; this is why the
most powerful instruments (including MRI instruments) use superconducting
magnets operating at ~4 K.
● The second factor that influences the ΔE between α and β states for a given
nucleus is the electron density near the nucleus; this is referred to as the shielding
of the nucleus.
Deshielded and Shielded Nucleii
Low electron density around a nucleus allows it to see most of the external field
(deshielded); while high electron density “masks” the nucleus from the field (shielded)
Deshielded and Shielded Nucleii (contd)
● Recall that the energy of a photon is directly related to its frequency (E = hν)
● Based on this, deshielded nucleii will require higher frequency radiation to
resonate; they will appear downfield in an NMR spectrum (to the left).
● Shielded nucelii will required lower frequency radiation to resonate; they will
appear upfield in an NMR spectrum (to the right).
1
H NMR Spectroscopy
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H (proton) NMR spectroscopy is the most common method: the nucleus gives a strong
signal and 1H is the most common isotope of hydrogen. Four types of information are
obtained from a 1H NMR spectrum:

1. The number of different protons in a molecule

2. The relative shielding/deshielding of each type of proton (chemical shift)
3. The number of protons contributing to each signal (integration)
4. The number of protons on carbon atoms adjacent to each signal (spin-spin
splitting; coupling constants)
Number of Different Protons (Signals)
● NMR can distinguish subtle differences in shielding, so that protons in different
positions will appear in different regions of the spectrum.
● However, protons in the same chemical environment will appear as the same
signal, even if they are spatially located on different positions on the molecule;
they are chemically equivalent.
Diastereotopic Protons
While homotopic and enantiotopic protons contribute to the same signal in 1H NMR,
diastereotopic protons are not chemically equivalent and will have separate signals.
Conformational Isomerism
● Internal rotations and ring flips occur
on a much faster time scale than the
interconversion between α and β spin
states for nuclei (NMR time scale).
● NMR signals for protons that
interconvert will appear as one signal.
Chemical Shift of Signals
Several factors impact the position of a signal in a 1H NMR spectrum, including the
degree of substitution of the carbon bearing the protons and the presence of electron
withdrawing atoms or functional groups
Chemical Shift and the ppm Scale
● Different strength magnets will give different frequency values for the same
protons in a molecule.
● Also, variations in field strength for the same model of magnet due to
manufacturing variations or age will lead to different frequencies.
● Because of this, the ppm scale (δ scale) was developed to standardize where a
signal appears in an NMR spectrum.
Chemical Shift and the ppm Scale (contd)
● Tetramethyl silane, (CH3)4Si (TMS) is
used as the reference standard, and
appears at 0 ppm.
● Most 1H NMR signals appear in the
range of δ = 0 - 10 ppm, though some
signals appear outside this range.
● Other nuclei (13C for example) use a
broader ppm scale.
Chemical Shift and the ppm Scale (contd)
This chart shows typical chemical shift ranges; note that some are quite broad.
Chemical Shift Ranges in 1H NMR Spectra
Bear in mind that deshielding effects are cumulative; this can make it challenging to “pin
down” signals based on chemical shifts. In general:

● Alkane signals show up at 0 - 2 ppm; primary < secondary < tertiary

● H’s on C’s next to a C=O, a C=C or aromatic ring appear at ~2 - 3 ppm
● H’s on C bearing a halogen or O appear at 3 - 5 ppm
● Alkene H signals show up at 5 - 7 ppm
● Aromatic H signals appear at 7 - 8 ppm
● Aldehyde H’s appear at 9 - 10 ppm
● Carboxylic acid H’s appear at 11 - 12 ppm
Ring Currents
π Systems generate a ring current when subjected to an external magnetic field. This
causes a magnetic field that either opposes (shielding) or supports (deshielding) protons,
depending on where they are positioned relative to the current.
Ring Currents (contd)
Alkene and aromatic protons are positioned in the deshielding portion of the ring
current; alkyne protons are in the shielding portion (δ ~ 2.4 ppm)
Integration of Signals
● The intensity of signals in 1H NMR
spectra are dependent on the
number of protons contributing to
the signal.
● Integration is the computer
processing signals converting the
area under each peak in the
spectrum into a vertical distance,
shown as an integral line
Integration of Signals (contd)
● More advanced spectrometers
typically just show the integration
number instead of the line.
● Integration does not show the total
number of protons for each signal;
what you see is the ratio of signal
areas.
● The 1:1.5 ratio here should be
interpreted as 2H:3H
Spin-Spin Splitting
● Protons in close proximity to the
proton that is resonating can be in
the α or β state; each of these states
will contribute differently to the
shielding effect.
● If a single proton is “next door”, the
ones in the β state will shield the
resonating proton more than the
ones in the α state; this will split the
resonating signal in two peak, a
doublet.
Spin-Spin Splitting (contd)
● In the example, the single proton in
the center splits the six methyl group
protons into a doublet.
● In the same manner, the six methyl
protons split the single proton into a
septet. There are seven possible
combinations of α and β spins for the
methyl protons.
● In general, n protons will cause n+1
splitting
Proton-Proton Exchange and Splitting
● The OH proton is relatively acidic, and
exchanges (goes off and on) with the
solvent for the sample.
● This exchange occurs rapidly versus
the interconversion rate between α
and β spin states. Result: no splitting.
● OH and NH protons do not cause
spin-spin splitting, and are not split
themselves. These protons often
show relatively broad peaks.
Common Splitting Patterns
The doublet/septet pattern seen before is
fairly common, indicating an isopropyl
group. Another common pattern is the
quarter/triplet splitting pattern of an ethyl
group.

There are other examples which you

should train yourself to recognize.
Coupling Constants
● Using a powerful spectrometer, it is possible to measure the difference in
frequency between the individual peaks in a split signal. This value is measured in
units of Hertz (Hz).
● This coupling constant allows the scientist to determine which signals are splitting
each other, and can simplify the structural assignment.
○ This is because the coupling constant match; if proton A’s signal is split by B
with a coupling constant of 4.3 Hz, B’s signal will be split by A with a 4.3 Hz
coupling constant.
● Coupling constants can also be used to resolve complex splitting patterns.
Complex Splitting Patterns
Often a proton will be split by
more than one set of equivalent
protons, leading to splitting
patterns that are more complex
than the n+1 rule. More powerful
NMR instruments are needed for
the resolution to interpret these
patterns.
13
C NMR Spectroscopy
The 13C nucleus can also be analyzed by NMR, but there are two problems:

1. The signal intensity for the 13C nucleus is much weaker than the 1H nucleus.
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2. C has a natural abundance of ~1%.
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C NMR had little practical application until the development of Free Induction Decay
(FID) methods coupled with the use of the Fourier Transform.
FID and FTNMR
In FID methods, rather than tuning the
radio frequency energy for a spectrum, the
sample is “blasted” with a broad spectrum
of radio waves, and the decay of the
resonance signal for all nuclei is then
observed. All the signals undergo decay,
and the overlapping decays appear as a
free induction decay plot.
Applying the Fourier Transform
The Fourier transform is a mathematical operation that converts the FID plot from a time
domain (x-axis) to a frequency domain.
How does FT-NMR Help for 13C?
● FID spectra are collected in a short period of time (the example was over 20 μsec).
Hundreds of spectra can be collected and averaged, greatly reducing the signal to
noise ratio.
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● C signals are further boosted by irradiating all the proton signals (broadband
decoupling); this prevents C/H spin-spin splitting (actually a good thing for 13C) and
increases the carbon signal through the Nuclear Overhauser Effect (NOE). The link
details the explanation, but you are not required to know how this works.
What Information is Obtained in 13C NMR?
● We do not obtain splitting patterns. It is unlikely that two 13C atoms will be
adjacent to each other, and NOE “kills” any C-H splitting.
○ If C-H splitting occurred, the spectrum would become very complex, and the
signal intensity would drop (split peaks are shorter than singlets).
● We do not obtain integrations; NOE boosts the signals of 13C atoms with more
attached protons over those with less or none.
● We do obtain information about the number of different carbon atoms and their
chemical shifts.
13
C NMR Sample Spectrum
Note that the carbonyl C (1) is the smallest
peak, not benefitting from NOE. Peaks 2
and 3 both are due to two equivalent C
atoms, while 4 is due to a single C.

The 13C NMR scale is much broader, with

signals in the range of δ = 0 - 240 ppm
13
C NMR
Chemical
Shift Ranges
Structure Determination from NMR Spectra
Tabular NMR Data
Often, instead of showing spectra, the information will be given in text or table form.
For the 1H and 13C spectra shown in the last slide:
1
H: δ = 2.45 (2H, q), 2.14 (3H, s), 1.06 (3H, t); 13C: δ = 209.3, 36.87, 29.43, 7.87

δ (ppm) splitting integration

δ = 209.3 ppm

2.45 quartet 2 δ = 36.87 ppm

2.14 singlet 3 δ = 29.43 ppm

1.06 triplet 3 δ = 7.87 ppm

Structural Elucidation
Frequently several different types of spectroscopy will be used to determine or verify the
identity of a compound:

● Mass spectrometry is used to determine the molecular weight (M+ ion), principal
fragmentation patterns (base peak) and the presence of some atoms (Cl, Br)
● IR spectroscopy is used to identify what functional groups are present.
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● H and 13C NMR spectroscopy can elucidate the molecule’s connectivity: how many
different types of H and C, certain functional groups, number of nearby H’s
● All these spectra are treated as “puzzle pieces”; the scientist needs to assemble
them to form a consistent picture.
Advanced NMR Experiments
● The use of computerized data
processing required for Fourier
transform also allows pulse
sequences that created more
sophisticated 13C spectra
● DEPT-135 is one example:
○ CH and CH3 are positive
○ CH2 is negative
2D NMR Experiments: COSY
● COSY plots the 1H NMR spectrum
on both axes; the spectrum
appears on the diagonal of the 2D
plot..
● Points off the diagonal show which
protons are coupled (splitting each
other)
2D NMR Experiments: COSY (contd)
● COSY (different link here) plots the
1
H NMR spectrum on both axes;
the spectrum appears on the
diagonal of the 2D plot..
● Points off the diagonal show which
protons are coupled (splitting each
other)
2D NMR Experiments: HETCOR
● HETCOR plots the 1H spectrum on
the x-axis and the 13C spectrum on
the y-axis.
● Points on the 2D plot show which
protons are attached to which
carbons.
Biochemical Applications of NMR
● NMR can be used to determine protein structure and protein behavior; it’s
advantage is that the protein can be analyzed in solution, where it assumes the
conformation it would have in its functional form.
● Other methods of structural analysis (specifically X-ray crystallography) require the
protein to be in solid, crystalline form. This form may not have the same shape
that the protein has in solution.
Magnetic Resonance Imaging (MRI)
● MRI uses the same principles as NMR, observing the protons in water in differing
biological environments.
● The data processing software associated with MRI interprets variations in the
proton signals to identify soft tissue structures. This make MRI complimentary to
X-rays, which looks at hard tissues.
● The name “MRI” was developed to address concerns about the word “nuclear” in
NMR.
Review Questions
1. Nuclei that can be analyzed by NMR spin to produce a net __________________.
2. In NMR, the resonance is the excitation/decay cycle between the ___________ and ________ states.
3. What type of EM radiation is required to induce resonance in a nucleus?
4. The presence of electrons near a nucleus decreases the effect of an external magnetic field in a
process called _______________; this decreases the energy required for resonance.
5. Nuclei that are close to electron-withdrawing atoms or groups appear ____________in NMR spectra.
6. Protons that are ______________ will contribute to the same NMR signal in a spectrum.
7. True or False: Enantiotopic protons have different signals in an NMR spectrum.
8. The position of a signal in an NMR spectrum is called its _______________, designated by δ.
9. Although differing positions in NMR spectra are based on resonance frequencies; the _______ scale is
used to account for differences in magnetic field strengths between instruments.
10. _______________ is used to measure the number of protons contributing to each NMR signal.
Review Questions (contd)
11. Aromatic protons are deshielded due to the _____________ of the π system in the molecule.
12. How many peaks will be seen for a signal split by four chemically equivalent protons?
13. OH and NH protons undergo ____________ due to their acidity; they do not participate in splitting.
14. It is possible to determine which protons split each other’s signals by measuring their _____________.
15. To compensate for the weak signal strength of 13C NMR, _________________spectra are collected,
averaged and converted from time to frequency domain using the _________________.
16. _________________ is the irradiation of proton signals to enhance carbon signals through NOE.
17. What two types of information collected in 1H NMR cannot be obtained in 13C NMR?
18. COSY and HETCOR are acronyms for two ________________ methods used to obtain more detailed
structural information on molecules.
19. NMR can be used for protein structural analysis; unlike x-ray crystallography, NMR does not require a
_______________ sample.