3/29/2016

Nuclear Magnetic Resonance Spectroscopy

(NMR)

Dr. Eddy Owaga

IFBT, DeKUT
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Overview
• Introduction
• Principle of NMR
• NMR instrumentation
• Advantages and Disadvantages
• Applications of NMR

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Definition of NMR Spectroscopy

• is a technique which exploits the magnetic properties of

certain atomic nuclei to study physical, chemical, and
biological properties of matter
• a powerful analytical technique used to characterize
organic molecules by identifying carbon-hydrogen
frameworks within molecules.

- NMR results from resonant absorption of electromagnetic

energy by a nucleus (mostly protons) changing its spin
orientation
- The resonance frequency depends on the chemical
environment of the nucleus giving a specific finger print of
particular groups (NMR spectroscopy)
- NMR is the phenomenon whereby a magnetic nuclei
absorbs and emits energy in the presence of a magnetic
field

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Types of NMR
• Two common types of NMR spectroscopy are
used to characterize organic structure:

– 1H NMR:- Used to determine the type and number of H

atoms in a molecule

– 13C NMR:- Used to determine the type of carbon atoms in

the molecule

Nuclear Spin
• A nucleus with an odd atomic number or an odd mass
number has a nuclear spin.
• The spinning charged nucleus generates a magnetic field

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Nuclear Spin

Nuclear Spin

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External Magnetic Field

When placed in an external field, spinning protons act like
bar magnets.

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Two Energy States

The magnetic fields of the

spinning nuclei will align
either with the external
field, or against the field.

A photon with the right

amount of energy can be
absorbed and cause the
spinning proton to flip.
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Effect of Magnetic field

A nucleus is in resonance when it absorbs radio frequency (RF)
radiation and “spin flips” to a higher energy state.

• Thus, two variables characterize NMR: an applied magnetic field

B0, the strength of which is measured in tesla (T), and the
frequency, v, of radiation used for resonance, measured in hertz
(Hz), or megahertz (MHz).

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NMR History
Nobel prizes
1944 Physics Rabi (Columbia) 1952 Physics Bloch (Stanford),
Purcell (Harvard)
"for his resonance
method for recording
the magnetic
properties of atomic
nuclei"

1991 Chemistry Ernst (ETH) "for their development

of new methods for
"for his contributions to nuclear magnetic
the development of the precision measurements
methodology of high and discoveries in
resolution nuclear connection therewith"
magnetic resonance
(NMR) spectroscopy"
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NMR History contd..

2002 Chemistry Wüthrich (ETH)

"for his development of nuclear magnetic

resonance spectroscopy for determining the
three-dimensional structure of biological
macromolecules in solution"

2003 Medicine Lauterbur (University of Illinois in

Urbana ), Mansfield (University of Nottingham)

"for their discoveries concerning

magnetic resonance imaging"

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Overview
• Introduction
• Principle of NMR
• NMR instrumentation
• Advantages and Disadvantages
• Applications of NMR

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Principle of NMR
 The sample is dissolved in a solvent, usually CDCl3(deutero-
chloroform) or Tetramethylsilane (TSM) , and placed in a
magnetic field.

 A radiofrequency generator then irradiates the sample with a

short pulse of radiation, causing resonance.

 When the nuclei fall back to their lower energy state, the
detector measures the energy released and a spectrum is
recorded.

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Principle of NMR(Contd…)

 Protons in different environments absorb at slightly different

frequencies, so they are distinguishable by NMR.

 The frequency at which a particular proton absorbs is

determined by its electronic environment.

 The size of the magnetic field generated by the electrons

around a proton determines where it absorbs.

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Principle of NMR(Contd…)
 Modern NMR spectrometers use a constant magnetic field
strength B0, and then a narrow range of frequencies (300-500 Hz)
is applied to achieve the resonance of all protons.

 Only nuclei that contain odd mass numbers (such as 1H, 13C, 19F
and 31P) or odd atomic numbers (such as 2H and 14N) give rise to
NMR signals.

The spin state of a nucleus is affected by an applied magnetic

18 field….

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Principle of NMR(Contd…)

absorb ∆E
α-spin states β-spin states
release ∆E

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Signals detected by NMR

at no magnetic field,
proton spin state
there is no difference beteen (higher energy)
- and - states.
Graphical relationship between
magnetic field (B o) and frequency (  E E = h x 300 MHz E = h x 500 MHz
1
for H NMR absorptions
proton spin state
(lower energy)

0T 7.05 T 11.75 T

Bo

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Overview
• Introduction
• Principle of NMR
• NMR instrumentation
• Advantages and Disadvantages
• Applications of NMR

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An NMR machine consists of:

(1) A powerful, supercooled magnet (stable, with sensitive
control, producing a precise magnetic field).
(2) A radio-frequency transmitter (emitting a very precise
frequency).
(3) A detector to measure the absorption of radiofrequency by
the sample.
(4) A recorder (to plot the output).

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Bench top NMR spectrophotometer

• Frequency:- 60 MHz
• Magnet:- Permanent electromagnet
• Available nuclei:- 1H, 19F
• Sample:- Standard 5mm NMR glass tubes
• Field strength:- 1.41T
• Resolution:- 70 ppb
• Operating temperature:- 18-26C
• Power supply:- 100-240 VAC(50-60 Hz)
• Dimensions:- 242843 cm
• Weight:- 18kg
• Software:- Mnova software

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Schematic of NMR Spectrometer

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The spectrometer

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Types of samples
• Both liquid and solid type of samples can be used in NMR
spectroscopy.

• For liquid sample, conventional solution-state NMR

spectroscopy is used for analysing where as for solid type
sample, solid-state spectroscopy NMR is used.

• In solid-phase media, samples like crystals, microcrystalline

powders, gels, proteins, protein fibrils or all kinds of polymers
etc. can be used.

• In liquid phase, different types of liquid solutions, nucleic acid,

protein, carbohydrates etc. can be used.
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NMR Signals

• The number of signals shows how many different kinds of

protons are present.
• The location of the signals shows how shielded or
deshielded the proton is.
• The intensity of the signal shows the number of protons of
that type.
• Signal splitting shows the number of protons on adjacent
atoms.

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The NMR Graph

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Chemical shift(Contd…)
The electrons surrounding a nucleus affect the effective
magnetic field sensed by the nucleus.

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Measurement of Chemical Shift

• Numeric value of chemical shift: difference between

strength of magnetic field at which the observed nucleus
resonates and field strength for resonance of a reference.

– Difference is very small but can be accurately measured

– Taken as a ratio to the total field and multiplied by 106 so

the shift is in parts per million (ppm)

• Absorptions normally occur downfield of Tetramethylsilane

(TMS), to the left on the chart.

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CH3
Tetramethylsilane
H3C Si CH3
CH3

• TMS is added to the sample.

• Since silicon is less electronegative than carbon, TMS

protons are highly shielded. Signal defined as zero.

• Organic protons absorb downfield (to the left) of the TMS

signal.

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Chemical Shift

• Measured in parts per million.

• Ratio of shift downfield from TMS (Hz) to total
spectrometer frequency (Hz).
• Same value for 60, 100, or 300 MHz machine.
• Called the delta scale.

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Location of Signals

• More electronegative atoms

deshield more and give larger shift
values.
• Effect decreases with distance.
• Additional electronegative atoms
cause increase in chemical shift.

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Chemical Shift Data

• different kinds of protons typically come at different chemical shifts.
• shown below is a chart of where some common kinds of protons appear
in the δ scale.
• Note that most protons appear between 0 and 10 ppm.
• The reference, tetramethylsilane (TMS) appears at 0 ppm, and aldehydes
appear near 10 ppm.

R
NH
R
OH

Ph R H
Me
OH
(R) TMS = Me Si Me
HO CH3 Ph CH3 Me
R R
R O R R
O NR2 O
Cl
H H H OCH3 CH3 CH3 CH3
R CH3
TMS

10 9 8 7 6 5 4 3 2 1 0
Downfield region ppm Upfield region
of the spectrum of the spectrum
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Interpreting 13C NMR

• number of different signals indicates the number of

different kinds of carbon

• location (chemical shift) indicates the type of functional

group.

• peak area indicates the numbers of carbons (if integrated).

• splitting pattern of off-resonance decoupled spectrum

indicates the number of protons attached to the carbon

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Two 13C NMR Spectra

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Typical Values

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1H NMR—The Spectrum

• NMR absorptions generally appear as sharp peaks.

• Increasing chemical shift is plotted from left to right.
• Most protons absorb between 0-10 ppm.
• The terms “upfield” and “downfield” describe the relative location of
peaks. Upfield means to the right. Downfield means to the left.
• NMR absorptions are measured relative to the position of a reference
peak at 0 ppm on the d scale due to tetramethylsilane (TMS). TMS is a
volatile inert compound that gives a single peak upfield from typical
NMR absorptions

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1H NMR—Number of Signals
• The number of NMR signals equals the number of different types of
protons in a compound.

• Protons in different environments give different NMR signals.

• Equivalent protons give the same NMR signal.

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• An NMR spectrum is a plot of the intensity of a peak against its chemical

shift, measured in parts per million (ppm).

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Overview
• Introduction
• Principle of NMR
• NMR instrumentation
• Advantages and Disadvantages
• Applications of NMR

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Advantages of NMR
- NMR is nondestructive and contact free
- Modern variants of NMR provide 3D structural resolution of
(not too large) proteins in solution
- Nuclear magnetic resonance spectroscopy basically provides
the detailed information about the structure, dynamics,
reaction state, and chemical environment of molecules.
- NMR is the most powerful tool available for organic structure
determination. It is used to study a wide variety of nuclei:
1H, 13C, 15N, 19F, 31P

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Disadvantages of NMR

• Any diamagnetic or paramagnetic ions present in the

formation can affect the tool response

• Expensive

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Overview
• Introduction
• Principle of NMR
• NMR instrumentation
• Advantages and Disadvantages
• Applications of NMR

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• Applications of NMR

energetic status of cells to

monitor the fermentation Analysis of sample quality
of yoghurts (using and in process control
phosphorus 31 LR NMR)

Applications

examination of cell
cooking of various types
cultures in the mashing of
of rice (proton NMR)
beer (using proton NMR)

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Carbohydrate NMR Spectroscopy

• structure of monosaccharides and oligosaccharides.

• sugar conformations.

• 500 MHz or greater.

• 3 – 6 ppm. (13C NMR chemical shifts of carbohydrate ring

carbons are 60 – 110 ppm).

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MRI
• Magnetic resonance imaging, noninvasive

• “Nuclear” is omitted because of public’s fear that it would be

radioactive.

• Only protons in one plane can be in resonance at one time.

• Computer puts together “slices” to get 3D.

• Tumors readily detected.

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- NMR tomography (Magnetic resonance imaging, MRI) is

the most advanced and powerful imaging tool
- It has various applications in food industries, food
science, chemical analysis of different products,
pharmaceutical approach etc.

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In summary…
• Introduction
• Principle of NMR
• NMR instrumentation
• Advantages and Disadvantages
• Applications of NMR

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