© 2005 Bastian Schirmer
English for Biochemistry, SS 2005
NMR spectroscopy
The parallels between human life and molecules are sometimes striking: The
Greek word άτομο (=atomos) not only means atom but “person” or
“individual”, too. Although no one ever thinks of a hydrogen atom as an
individual, in fact the chemical behaviour of an atom depends on its
surroundings and different atoms in a molecule can be distinguished just by
taking a closer look at the atoms and their “social contacts”. By means of NMR
spectroscopy this information about individual atoms and their neighbours in
molecules can be gained easily.
NMR is the abbreviation for “nuclear magnetic resonance”, which implies that
the possibility to detect nuclei with NMR spectroscopy is closely linked to their
magnetic properties. Thus it is very important to know about the origin of
nuclear magnetism. Many nuclei behave as if spinning around their own axis.
This rotation can be described by the spin angular momentum I, which is a
quantized vector quantity. The magnitude of the spin angular momentum
directly corresponds to the nuclear spin number I (magnitude of I =
[h√I(I+1)]/2), which usually adopts values between I=0 and I=4. Every proton
and neutron possesses a spin I=1/2 and therefore these particles are often called
the “spin-1/2” particles. The spin of a proton can only pair up with another
proton’s spin, but not with a neutron’s and vice versa. Therefore nuclei with
even numbers of protons and neutrons have no nuclear spin since all protons
and neutrons are spin-paired. Nuclei with both numbers of protons and
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neutrons being odd have an integral positive spin and nuclei with only one of
the particle numbers being odd exhibit a spin which is an odd integer multiple
of ½. In addition there is a magnetic quantum number m, which can adopt 2I
+ 1 values ranging from -I to +I in integral steps. The multiplicity (2I + 1) tells
us about the number of possible (and degenerate) spin states because there are
only mh/2 projections of the spin angular momentum I onto an axis chosen at
random. If an external magnetic field is applied this axis is the one parallel to
the field. A very lucid explanation for using the projection of I is the spin
angular momentum’s precession around the projection axis in a magnetic field:
The net spin angular momentum coincides with the projection of I onto the
axis. The existence of a net spin angular momentum gives rise to a magnetic
moment  which is directly proportional to I (I) and hence can be parallel
or sometimes antiparallel to the spin angular momentum. The proportionality
constant  is called the magnetogyric ratio and depends only on the nucleus
being examined. Exposing a nucleus to an external magnetic field puts an end
to the degeneracy of possible spin orientations. Now the differently orientated
magnetic moments even have different energies: E = -(B stands for the
strength of the magnetic field in Tesla). In order to induce a transition between
the spin states (only transitions with m = ±1 are allowed) radiofrequency
radiation of a certain energy quantum is required. The corresponding frequency
is called the resonance frequency, which fulfills the Planck resonance condition
E = hhB/2 Note that the strength of the external field influences the
extent of the energy gap between the spin states. The stronger the magnetic
field, the larger the energy gap.
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In NMR spectrometers there is a transmitter coil which sends radiofrequency
radiation into the sample and a receiver coil which detects the radiation left
after having passed through the sample. The sample substance has to be
dissolved in a solvent which does not absorb frequencies from the spectral area
being observed. Now the resonance frequency can be identified as the
frequency absorbed by the nucleus. The array of transmitter coil, sample, and
receiver coil is embedded in a superconducting magnet cooled down by liquid
helium and nitrogen. The sample tube is set into rotation by a continuous
stream of compressed air in order to average all possible molecule orientations.
Because of the unique magnetogyric ratio of different nuclei each nucleus
absorbs a characteristic energy and thus can be detected by NMR ― provided
that the nucleus possesses a nuclear spin. If the nuclear spin is zero there is no
magnetic moment either so that different nuclear spin energy levels in a
magnetic field cannot exist.
But NMR can do much more than just identify nuclei in molecules. Depending
on its surroundings in a molecule a nucleus can adopt several slightly different
resonance frequencies. This phenomenon is called chemical shift and is due to
the local electronic properties of nuclei in the molecule examined. Magnetic
fields induce a circular motion of electrons in their orbitals causing a local
magnetic field which is at its origin opposed to the external field. Thus the
nucleus is shielded from the magnetic field and has a higher resonance
frequency. Several factors, such as the geometry of molecular orbitals,
inductive, and mesomeric effects, influence the amount of shielding. If electron
density is decreased by mesomeric or inductive effects the nucleus is
deshielded. If it is increased the nucleus will be shielded. Aromatic molecule
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systems demonstrate the role of molecular orbital geometry in NMR
spectroscopy: These cyclic structures show a continuous circular distribution of
electrons above and below the aromatic ring. The hydrogen atoms on the
periphery of the molecule are deshielded because the induced local magnetic
field opposes the external field only in the very center of the ring but augments
it on the periphery. In order to measure the chemical shift a standard has to be
determined. The difference between the standard’s and the nucleus’s resonance
frequency gives rise to a dimensionless parameter called 106 (/ref),
which, in fact, is a quantity of deshielding. The commonly used standard for
13
C or 1H-NMR is tetramethylsilane (CH3)4Si and its signal is situated in the
right corner of the spectrum, which means that the value of  grows larger
from right to left. Every signal peak in an NMR spectrum refers to the
standard. When dealing with an NMR spectrum there will not always be such a
simple structure of peaks. In most cases signal multiplets can be observed
because magnetic nuclei interact with others in their immediate vicinity. This is
called scalar or spin-spin coupling and leads to split signal peaks. As a rule of
thumb a nucleus with N non equivalent nuclei in immediate vicinity shows a
signal split into N+1 peaks. The term “equivalent” here means that both nuclei
are situated in identical environments and hence possess the same chemical
shift. The main criterion for the equivalence of nuclei is the symmetry of
molecules: Nuclei in symmetrical positions within the molecule are equivalent.
After having attained structural information on the molecule even quantitative
aspects can be investigated. The surface area of the peaks indicates the relative
amount of corresponding nuclei in a molecule.
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These fundamental approaches to determine the structure of a molecule have
shown the performance of NMR techniques themselves. But how can those
basic techniques be applied to the problems of daily laboratory work? There
are different types of NMR spectroscopy serving various purposes. For a long
time scientists used continuous wave NMR. A certain frequency of radiation
was emitted into the sample and the strength of the magnetic field was varied
in order to obtain a complete NMR spectrum of the sample. Nowadays
radiofrequency pulse techniques are preferred because of their higher
sensitivity and their speed. The radiofrequency pulse emitted is very short and
covers a large spectrum of frequencies. Thus it is not necessary to search
slowly for the nuclei’s resonance frequencies because they will all be excited at
once. That means, in fact, a single spectrum is obtained much faster. The time
saved can be used to improve the sensitivity by recording several spectra and
coadding them so that most of the noise will vanish. These advantages require
some technical improvements such as radiofrequency amplifier, pulse
coordinator, and a powerful computer. In the beginning the spectrum is
generated time-dependently. The computer performs the task of Fourier
transformation in order to obtain the common NMR spectrum. Instead of
working with just one single pulse it sometimes is more appropriate to use
pulse sequences. Two-dimensional NMR methods such as Nuclear Overhauser
Effect spectroscopy (NOESY) or correlated spectroscopy (COSY) benefit from
this practice and have enhanced the effectiveness of NMR. It can be stated that
pulse methods have widened the range of NMR applications: Metabolic
pathways and microbiological structures can be observed, proteins and other
macromolecules as well as the tissues of human body can be explored. The
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benefits of NMR are not solely used by chemists and physicists any more but
by life scientists, physicians, and biochemists, as well.
Prof. Ray Freeman from University of Cambridge once compared NMR with
espionage: The atoms themselves are the spies scientists can use to gain
structural information on molecules. NMR is a powerful instrument to
investigate (new) molecules, because nearly every nucleus is an “individual”.
The “social contacts” of atoms do not only bond them together but betray them
to the scientist who profits from this perfidious submicroscopic society.
References:
[1]
Krishna, N. R., Berliner, Lawrence J., Eds., Biological Magnetic
Resonance, Volume 20: Protein NMR for the Millennium, Kluwer
Academic / Plenum Publishers, New York, 2003
[2]
Barbotin, Jean- Noël, and Portais, Jean-Charles, Eds., NMR in
Microbiology. Theory and Applications, Horizon Scientific Press,
Wymondham, 2000
[3]
Hore, P. J., Nuclear Magnetic Resonance, Oxford University Press,
New York, 1995
[4]
Macomber, Roger S., A complete introduction to modern NMR
spectroscopy, Wiley, New York, 1998
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[5]
Vollhardt, K. P. C., Schore, Neil E., Organische Chemie, 3rd edition,
Wiley-VCH, Weinheim, 2000
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