COURSE#1022: Biochemical Applications of NMR Spectroscopy

http://www.bioc.aecom.yu.edu/labs/girvlab/nmr/course/

Basic Principles

LAST UPDATE: 1/19/2007

Reading
Selected Readings for Basic Principles of NMR:
Evans, pp 2-13
Cavanagh et al, pp 1-18
Derome, pp 63-76
Macomber, Chapters 1 and 2

Online Sources
Content from the following online sources were used for some slides in this
presentation:
http://tonga.usip.edu/gmoyna/NMR_lectures
http://www.oci.unizh.ch/group.pages/zerbe/notes.html
http://www.cis.rit.edu/htbooks/nmr/

What is Spectroscopy?
Spectroscopy is the study of the interaction of light with matter.
Here light refers to any sort of electromagnetic radiation, such as visible light,
UV, IR, microwaves and radiowaves.
Depending on the frequency or wavelength of the radiation involved we will
have different types of interactions with matter (molecules).
The following chart shows the ranges (wavelengths), for different types of
spectroscopies.
-rays

10-10

x-rays

10-8

10-6

UV VIS

IR

10-4
10-2
wavelength (, cm)

-wave

100

radio

102

wavelength and frequency are inversely proportional, so higher frequencies

mean shorter wavelength.

Nuclear magnetic resonance (NMR) spectroscopy is the absorption of

radio frequencies by atomic nuclei within a sample that is placed in a magnetic
field. The types of sample that can be studied are liquids, solids and whole
organisms! NMR spectroscopy finds applications in several areas of science and
is routinely used to study chemical and biochemical structure and function using
simple one-dimensional techniques and more complicated multidimensional
techniques.
A typical simple Fourier Transform (FT) NMR experiment involves:
put sample (g to g quantities) into a magnet
excite nuclei with radiofrequencies using RF probe
record nmr spectrum of RF absorbed by sample (seconds to days)

nmr spectrum
magnet

RF probe

Spin
An atomic nucleus is a collection of protons and neutrons which, like electrons,
possess a quantum mechanical property called spin which is characterized by an
intrinsic spin angular momentum. Spin angular momentum is an intrinsically
quantum mechanical property that does not have a classical analog. Spin is a
fundamental property of nature like electrical charge or mass. All of these
particles are spin=1/2 particles. The nucleus itself has a total spin angular
momentum, I, formed by the coupling of the individual spin angular momenta of
its constituent protons and neutrons (nucleons). The total nuclear spin angular
momentum quantum number may therefore take values: 0, 1/2, 1, 3/2, 2, 5/2,
etc.

Spin and Nuclear Magnetic Moment

In physics, the magnetic moment is a measure of the strength of a magnetic
source.
Each nuclear spin has a magnetic moment which is associated with the
angular momentum of the nucleus. When any charged particle is rotating, it
behaves like a current loop with a magnetic moment. The nucleus has a
positive charge and is spinning. This generates a small magnetic field. The
nucleus therefore possesses a magnetic moment, , which is proportional to
its spin, I

- The constant, , is called the magnetogyric ratio and is a fundamental

nuclear constant which has a different value for every nucleus.
- h is Planks constant and has a value of 6.626 x 10-34 J s (in kms units).

Nuclear spin may be related to the nucleon composition of a nucleus

in the following manner:
Odd mass nuclei (i.e. those having an odd number of nucleons)
have fractional spins. Examples are I = 1/2 (1H, 13C, 15N, 19F, 31P), I =
3/2 (11B) and I = 5/2 (17O ).
Even mass nuclei composed of odd numbers of protons and
neutrons have integral spins. Examples are I = 1 (2H, 14N).
Even mass nuclei composed of even numbers of protons and
neutrons have zero spin ( I = 0 ). Examples are 12C, and 16O.
Spin 1/2 nuclei have a spherical charge distribution, and their nmr
behavior is the easiest to understand. Other spin nuclei have
nonspherical charge distributions and may be analyzed as prolate or
oblate spinning bodies. All nuclei with non-zero spins have magnetic
moments (), but the nonspherical nuclei also have an electric
quadrupole moment (eQ). Some characteristic properties of selected
nuclei are given in the following table.

Magnetic Properties of Some Useful Nuclei

Isotope

Natural
Abundance
(%)

Spin (I)

Magnetogyric
Ratio ()
107 rad s-1 T-1

Absolute
Sensitvity

1H

99.98

1/2

26.752

1.00

2H

0.02

4.107

1.45x10-6

3H

0.00

1/2

28.535

12C

98.90

13C

1.11

1/2

6.728

1.76x10-4

14N

99.63

1.934

1.01x10-3

15N

0.37

1/2

-2.712

3.85x10-6

16O

99.76

17O

0.04

5/2

-3.628

1.08x10-5

19F

100.0

1/2

25.181

0.83

31P

100.0

1/2

10.841

0.066

It is important to note that the 12C isotope of carbon and the 16O isotope of oxygen
have a spin of 0 this means that the main building blocks of organic compounds
cannot be observed by NMR spectroscopy. NMR studies of organic compounds
make use of the natural abundance of 13C for carbon NMR. Isotopic labeling of
compounds (eg. 13C replaces 12C) can be used under conditions where the
natural abundance content is insufficient such as in biomolecules.
Also, for most nuclides the nuclear angular momentum vector and the magnetic
moment vector point in the same direction, i.e. they are parallel and have a
positive magnetogyric ratio. However, in a few cases, for example 15N and 17O
(and also the electron), they are antiparallel and have a negative magnetogyric
ratio. The consequences of this condition will be considered later.

http://www.bruker.de/guide/eNMR/chem/NMRnuclei.html
Group

II

IIIa

IVa

Va

VIa

VIII
a

VIIa

VIII
b

VIII
c

IB

IIB

III

IV

VI

VII

VIII

Period
1

1
H

2
He

3
Li

4
Be

5
B

6
C

7
N

8
O

9
F

10
Ne

11
Na

12
Mg

13
Al

14
Si

15
P

16
S

17
Cl

18
Ar

19
K

20
Ca

21
Sc

22
Ti

23
V

24
Cr

25
Mn

26
Fe

27
Co

28
Ni

29
Cu

30
Zn

31
Ga

32
Ge

33
As

34
Se

35
Br

36
Kr

37
Rb

38
Sr

39
Y

40
Zr

41
Nb

42
Mo

43
Tc

44
Ru

45
Rh

46
Pd

47
Ag

48
Cd

49
In

50
Sn

51
Sb

52
Te

53
I

54
Xe

55
Cs

56
Ba

71
Lu

72
Hf

73
Ta

74
W

75
Re

76
Os

77
Ir

78
Pt

79
Au

80
Hg

81
Tl

82
Pb

83
Bi

84
Po

85
At

86
Rn

87
Fr

88
Ra

**

103
Lr

104
Unq

105
Unp

106
Unh

107
Uns

108
Uno

109
Mt

110
Uun

111
Uuu

112
Uub

113
Uut

114
Uuq

115
Uup

116
Uuh

117
Uus

118
Uuo

*Lanthanides

57
La

58
Ce

59
Pr

60
Nd

61
Pm

62
Sm

63
Eu

64
Gd

65
Tb

66
Dy

67
Ho

68
Er

69
Tm

70
Yb

**Actinides

**

89
Ac

90
Th

91
Pa

92
U

93
Np

94
Pu

95
Am

96
Cm

97
Bk

98
Cf

99
Es

100
Fm

101
Md

102
No

5/2

7/2

Nuclear Spins

1/2

3/2

9/2

Quantization of Spin
The magnitude of the spin angular momentum vector is

Where = h/2 and h is Planks constant, the unit of quantization and has a
value of 6.626 x 10-34 J s (in kms units). This angular momentum is spacequantized: it can only adopt 2I+1 orientations with respect to an arbitrary axis
(usually taken to be the z-axis). That is, the projection of I onto the z-axis is given
by

m is magnetic quantum number.

Spin States of I=1/2

For example, a nucleus (i.e. a proton), with I=1/2 is described as having two states
defined by m=+1/2 (spin up or state) and m=-1/2 (spin down or state).

The nuclear spin states of a hydrogen nucleus: the nuclear magnetic angular
momentum vector, I makes a projection onto the z-axis of (+1/2)h/2 or
(-1/2)h/2. In the absence of an applied magnetic field, these states are
degenerate.

Spin States of I=1

For spins with I=1 nuclei three different values for Iz are allowed (m=1,0,-1):

Iz = +h/2

Iz = 0

Iz = -h/2

Effect of a Magnetic Field on I=1/2

In the ground state all nuclear spins are disordered, and there is no energy
difference between them. They are degenerate:

= h / 4

Since they have a magnetic moment, when nuclear spins are placed in a strong
external magnetic field (Bo), they orient either against or with it:

Bo

There is always a small excess of nuclei (population excess) aligned with the field
than pointing against it.

Energy of I=1/2 in a Magnetic Field

The aligned nuclei can occupy two energy states, depending on which direction
they align in the magnetic field (parallel or antiparallel). The potential energy of an
magnetic moment in an external field is given by:

mI=+1/2 for parallel alignment with external field

mI=-1/2 for antiparallel alignment with external field
The later stage represents a energetically higher configuration.

Selection Rule and Transitions

Spins can undergo a transition between two energy states by the absorption of a
photon. The quantum-mechanical selection rule states that only transitions with
m=1 are allowed.

The energy of this photon must exactly match the energy difference between the
two states. The energy, E, of a photon is related to its frequency, , by Plank's
constant (h = 6.626x10-34 J s).

E=h and E = hB/2 so =B/2

In NMR, the quantity is called the resonance frequency or the Larmor
frequency.

k is Boltzmann's constant, 1.3805x10-23 J/Kelvin

T is the temperature in Kelvin.

Classical Mechanics and the Vector Model

The NMR sample is an ensemble of many nuclear spins and we can understand
much about the NMR experiment by examining the behavior of bulk
magnetization resulting from the ensemble using classical mechanics.

This represents the situation in most real samples. However, there is an

important difference between and M0. The former is quantized and can only be
in one of two states ( or ) while M0 tells about the whole spin population.

When a nuclear magnetic moment is placed in a magnetic field B it

experiences a torque tending to turn it parallel to the field direction.
Mathematically this is described by forming the vector product between dipole
moment and magnetic field:
d/dt = x B
This causes it to precess around the field (like a gyroscope under the
influence of gravity. The angular frequency of precession, 0, is proportional
to the field, B. At thermal equilibrium the spins precess in the magnetic field
but they have random phases- no phase coherence.

The Bloch Equations

The Bloch equations are a set of coupled differential equations which were
originally proposed by Felix Bloch to describe the motion of the net magnetic
moment, M, under different conditions. When properly integrated, the Bloch
equations will yield the X, Y, and Z components of magnetization as a function
of time.
The full Bloch equations contain relaxation terms (to be discussed in a future
class) that drive the system back to the equilibrium state (no transverse
coherence by T2 relaxation and relative population of the / states according
to the Boltzmann distribution by T1 relaxation).

In an NMR instrument, the detector is in the transverse plane perpendicular to

the Bo magnetic field. In order to detect the magnetization within the sample, we
must tip it from the z-axis into the transverse plane. From the Bloch equations,
in order to change the magnetization along the z-axis (dMz/dt)

a magnetic field has to be introduced along the x- or y-axis (Bx or By,- also
known as a B1 field). Since the magnetization is precessing at the Larmor
frequency, transverse magnetic fields that rotate about Z at the same frequency
will create a constant torque on the spins and rotate them away from the z-axis.

The NMR Spectrum

The spectrum consists of signals that have the following properties:
position or chemical shift
intensity which is proportional to # of nuclei
width which is sensitive to dynamics and size of molecule
splittings or couplings which is sensitive to # of bonded nuclei
So the signals within an NMR spectrum depend on the surrounding
environment (i.e. structure) and dynamics of the molecule

1H

NMR spectrum of an organic compound

1H

NMR spectrum of a protein

MKS units and Useful Constants

Unit

Variable

Symbol

MKSA units

Ampere

Current

fundamental MKSA unit

Coulomb

Charge

Ampere second

Farad

Capacitance

Coulomb Volt-1 = Joule Volt-2

Henry

Inductance

Weber Ampere-1 = Tesla meter2 Ampere-1

Joule

Energy

Newton meter = kilogram meter2 second-2

Kelvin

Temperature

fundamental MKSA unit

kilogram

Mass

fundamental MKSA unit

meter

Length

fundamental MKSA unit

Newton

Force

kilogram meter second-2

Ohm

Resistance

Volt Ampere-1 = Volt2 Joule-1 second-1

Pascal

Pressure

Newton meter-2

second

time

fundamental MKSA unit

Siemens

conductance

Ampere Volt-1 = Joule second Volt-2

Tesla

Magnetic field

Newton Ampere-1 meter-1 = Joule Ampere-1 meter-2 = Volt second meter-2

Volt

potential

Joule coulomb-1

Watt

Power

Joule second-1

Weber

Magnetic flux

Tesla meter2 = Volt second

k
Boltzmann's constant
1.3805 x 10-23 J/Kelvin
h
Planks constant
6.626 x 10-34 J s
-1
(rad/s) = 2 (s )
104 Gauss = 1 Telsa The earth's magnetic field in Rochester, New York is approximately 5x10-5 T.
= 3 x 108 m/s
1 cal = 4.18 Joules