SPECIAL TOPICS IN ANALYTICAL CHEMISTRY
Introduction to NMR
CHEM 921, Fall 2005
MWF 11:30-12:20pm, Rm 548 Hamilton Hall
COURSE OUTLINE
Instructor: Dr. Robert Powers
Office
Address: 722 HaH
Phone: 472-3039
Labs
720 HaH
472-5316
Office Hours: 10:30-11:30 am MWF or by Special Appointment.
web page: http://bionmr-c1.unl.edu
Text: “NMR and Chemistry: An Introduction to Modern NMR Spectroscopy ”, by J. W.
Akitt & B. E. Mann; Stanley Thornes, 2000
Course Work:
Written Report:
Exam 1:
Exam 2:
Structure Problems (5)
Total:
50 pts
100 pts
100 pts
150 pts
400 pts
(Fri., Sept. 9th)
(Fri., Sept 23rd)
(Wed., Nov. 2nd)
(as assigned)
PAPER ON NMR METHOD
• Paper General
– >2-3 pages single space text
• Additional pages for figures, references
– 12 pitch font, 1” margins
– Double spacing between paragraphs and headings
• Paper Topic
– Review a Manuscript that Describes an NMR Technique useful
for the Structural Analysis of a Chemical Structure
• The paper should not cover a method discussed in class or in the
text book
• The paper can describe a new modification or an improvement of a
method discussed in class or in the text book
PAPER ON NMR METHOD
• When Writing your Review Consider the Following:
•
•
•
•
•
•
Principals/theory behind technique
Issue or problem the technique is addressing
How does the new technique compare to existing approaches?
How the technique is used
What results were achieved?
Advantages/disadvantages
• Recommended Journals:
– Journal of Magnetic Resonance, Journal of Biomolecular NMR,
Magnetic Resonance in Chemistry, Magnetic Resonance
Quarterly, and Progress in NMR Spectroscopy
• Grading (50 points total)
• Due Date: 11:30 am, Friday Sept. 9th
STRUCTURE PROBLEMS
• Using NMR Data to Determine the Structure of Unknown
Organic Compound
• Total of 5 unknown structures to solve
• Structures will be randomly assigned one week before due date
• NMR data sets for unknowns will be available in the NMR computer
lab (832 HaH)
• Each unknown will include the following data:
– Chemical formula
– 1D 1H & 13C spectra
– 2D 1H COSY, 2D 1H NOESY, 2D 1H-13C HMQC, 2D 1H-13C HMBC
• Software Training
• You will need to know how to use Bruker’s TopSpin/XWinNMR
software to analyze the NMR data.
• Schedule a Training time
• Dr. Dumais (834 HaH,472-6255) and Ms. Basiaga (832B,472-3797)
will oversee training and scheduling of the NMR computer lab.
• A sample data set will be available for practice. Please use this
opportunity to familiarize yourself with the software ASAP.
STRUCTURE PROBLEMS
• Completed Reports for the Unknown Structure
Determination will contain:
• Unknown Number
• Chemical Structure of the Unknown
• 1H and 13C chemical shift Assignments
• Summary of Key Experimental Information that Support the
Structure
STRUCTURE PROBLEMS
• The Goal of these Problem Sets is to Develop Your Skills
in Analyzing NMR Data
•
•
•
•
These are individual projects
Do Not Work Together on the Assignments
Do Not Share Your Results with Other Students
Graded Problem Sets will be Returned After all Five Problem Sets
have been Completed
• The Due Dates for the Structure Problem Sets are:
•
•
•
•
•
Structure Problem #1 Fri. Sept. 30th
Structure Problem #2 Fri. Oct. 7th
Structure Problem #3 Fri. Oct. 14th
Structure Problem #4 Fri. Oct. 21st
Structure Problem #5 Fri. Oct. 28th
• Grading (30 points/structure; 150 total points)
Lecture Topics
I.
II.
III.
IV.
Topic
Chapters in NMR & Chemistry
BASIC NMR Theory
I.
Introduction to NMR Theory
1
a. Quantum and classical description
II. Obtaining an NMR Spectra
5
a. Data acquisition and processing
b. Instrumentation
III. Chemical Shifts (d)
2
a. Select examples of chemical shift trends
b. Predicting chemical shifts
IV. Coupling Constants (J)
3
a. Simulation of second-order spin systems
One and Two Dimensional NMR
I.
NMR Pulses
6
II. 1D NMR
8
a. NOE, J modulation, INEPT, DEPT, INADEQUATE
III. 2D NMR
9
a. Theory
b. COSY,TOCSY,NOESY,HMQC,HMBC
IV. Examples of Spectral Interpretation
8
NMR Dynamics
I.
Relaxation
4
a. T1,T2,Dipole-Dipole,CSA,Quadrupolar
II. Exchange
7
a. NMR time scale
Solid State NMR (as time permits)
11
Some Suggested NMR References
>>> “Spin Dynamics – Basics of Nuclear Magnetic Resonance” M. H. Levitt <<<
>>> “Structure Determination of Organic Compounds: Tables of Spectral Data” <<<
Pretsch, E., Bühlmann, P., and Affolter, C.
“Spectrometric Identification of Organic Compounds” Silverstein, Bassler and Morrill
“Basic One- and Two-Dimensional NMR Spectroscopy” Horst Friebolin
“Modern NMR Techniques for Chemistry Research” Andrew E. Derome
“Nuclear Magnetic Resonance Spectroscopy” R. K Harris
“Protein NMR Spectroscopy: Principals and Practice”
John Cavanagh, Arthur Palmer, Nicholas J. Skelton, Wayne Fairbrother
“Biomolecular NMR Spectroscopy” J. N. S. Evans
“NMR of Proteins and Nucleic Acids” Kurt Wuthrich
Some NMR Web Sites
The Basics of NMR by J.P. Hornak Hypertext based NMR course
http://www.cis.rit.edu/htbooks/nmr/nmr-main.htm
Integrated Spectral Data Base System for Organic Compounds
http://www.aist.go.jp/RIODB/SDBS/menu-e.html
Educational NMR Software
All kinds of NMR software
http://www.york.ac.uk/depts/chem/services/nmr/edusoft.html
NMR Knowledge Base
http://www.spectroscopynow.com/
A lot of useful NMR links
NMR Information Server
http://www.spincore.com/nmrinfo/
News, Links, Conferences, Jobs
Technical Tidbits
http://www.acornnmr.com/nmr_topics.htm
Useful source for the art of shimming
BMRB (BioMagResBank)
http://www.bmrb.wisc.edu/
Database of NMR resonance assignments
NMR History
1937
1946
1953
1966
1975
1985
Rabi predicts and observes nuclear magnetic resonance
Bloch, Purcell first nuclear magnetic resonance of bulk sample
Overhauser NOE (nuclear Overhauser effect)
Ernst, Anderson Fourier transform NMR
Jeener, Ernst 2D NMR
Wüthrich first solution structure of a small protein (BPTI)
from NOE derived distance restraints
1987
3D NMR + 13C, 15N isotope labeling of recombinant proteins
(resolution)
1990
pulsed field gradients (artifact suppression)
1996/7 new long range structural parameters:
- residual dipolar couplings from partial alignment in liquid
crystalline media
- projection angle restraints from cross-correlated relaxation
TROSY (molecular weight > 100 kDa)
Nobel prizes
1944 Physics Rabi (Columbia)
1952 Physics Bloch (Stanford), Purcell (Harvard)
1991 Chemistry Ernst (ETH)
2002 Chemistry Wüthrich (ETH)
2003 Medicine Lauterbur (University of Illinois in Urbana ),
Mansfield (University of Nottingham)
NMR History
First NMR Spectra on Water
1H
NMR spectra of water
Bloch, F.; Hansen, W. W.; Packard, M. The nuclear induction experiment.
Physical Review (1946), 70 474-85.
NMR History
First Observation of the Chemical Shift
1H
NMR spectra ethanol
Modern ethanol spectra
Arnold, J.T., S.S. Dharmatti, and M.E. Packard, J. Chem. Phys., 1951. 19: p. 507.
Nuclear Magnetic Resonance
Introduction:
Nuclear Magnetic Resonance (NMR) measures the absorption of electromagnetic
radiation in the radio-frequency region (~4-900 MHz)
- nuclei (instead of outer electrons) are involved in absorption process
- sample needs to be placed in magnetic field to cause different energy
states
NMR was first experimentally observed by Bloch and Purcell in 1946 (received Nobel
Prize in 1952) and quickly became commercially available and widely used.
Probe the Composition, Structure, Dynamics and Function of the Complete Range of
Chemical Entities: from small organic molecules to large molecular weight polymers and
proteins.
NMR is routinely and widely used as the preferred technique to rapidly elucidate the
chemical structure of most organic compounds.
One of the MOST Routinely used Analytical Techniques
O
Typical Applications of NMR:
1.) Structural (chemical) elucidation
 Natural product chemistry
 Synthetic organic chemistry
- analytical tool of choice of synthetic chemists
- used in conjunction with MS and IR
2.) Study of dynamic processes
 reaction kinetics
 study of equilibrium (chemical or structural)
3.) Structural (three-dimensional) studies
 Proteins, Protein-ligand complexes
 DNA, RNA, Protein/DNA complexes
 Polysaccharides
4.) Drug Design
 Structure Activity Relationships by NMR
5) Medicine -MRI
MRI images of the Human Brain
O
O
NH
O
O
OH
O
OH
HO
O
O
O
O
O
Taxol (natural product)
NMR Structure of MMP-13
complexed to a ligand
Each NMR Observable Nuclei Yields a Peak in the Spectra
“fingerprint” of the structure
2-phenyl-1,3-dioxep-5-ene
1H
NMR spectra
13C
NMR spectra
Information in a NMR Spectra
g-rays x-rays UV VIS
1) Energy E = hu
h is Planck constant
u is NMR resonance frequency 10-10
Observable
Name
10-8
IR
m-wave radio
10-6 10-4
10-2
wavelength (cm)
Quantitative
100
102
Information
d(ppm) = uobs –uref/uref (Hz)
chemical (electronic)
environment of nucleus
peak separation
(intensity ratios)
neighboring nuclei
(torsion angles)
Peak position
Chemical shifts (d)
Peak Splitting
Coupling Constant (J) Hz
Peak Intensity
Integral
unitless (ratio)
relative height of integral curve
nuclear count (ratio)
T1 dependent
Peak Shape
Line width
Du = 1/pT2
peak half-height
molecular motion
chemical exchange
uncertainty principal
uncertainty in energy
A Basic Concept in ElectroMagnetic Theory
A Direct Application to NMR
A moving perpendicular external
magnetic field will induce an
electric current in a closed loop
An electric current in a closed
loop will create a perpendicular
magnetic field
A Basic Concept in ElectroMagnetic Theory
For a single loop of wire, the magnetic field, B
through the center of the loop is:
B
mo I
2R
mo – permeability of free space (4p x 10-7 T · m / A)
R – radius of the wire loop
I – current
A Basic Concept in ElectroMagnetic Theory
Faraday’s Law of Induction
• If the magnetic flux (FB) through an area bounded by a closed conducting loop
changes with time, a current and an emf are produced in the loop. This process is
called induction.
• The induced emf is:
dF B
 
dt
Simple AC generator
A Basic Concept in ElectroMagnetic Theory
Lenz’s Law
• An induced current has a direction such that the magnetic field of the current
opposes the change in the magnetic flux that produces the current.
• The induced emf has the same direction as the induced current
Direction of current
follows motion of magnet
Theory of NMR
Quantum Description
Nuclear Spin (think electron spin)
•
Nucleus rotates about its axis (spin)
•
Nuclei with spin have angular momentum (p) or spin
1) total magnitude
 I ( I  1)
l
1) quantized, spin quantum number I
2) 2I + 1 states:
I=1/2:
I, I-1, I-2, …, -I
-1/2, 1/2
3) identical energies in absence of external magnetic field
NMR Periodic Table
NMR “active” Nuclear Spin (I) = ½:
1H, 13C, 15N, 19F, 31P
biological and chemical relevance
Odd atomic mass
I = +½ & -½
NMR “inactive” Nuclear Spin (I) = 0:
12C, 16O
Even atomic mass & number
Quadrupole Nuclei Nuclear Spin (I) > ½:
14N, 2H, 10B
Even atomic mass & odd number
I = +1, 0 & -1
Magnetic Moment (m)
•
spinning charged nucleus creates a magnetic field
Magnetic moment
Similar to magnetic field
created by electric current
flowing in a coil
“Right Hand Rule”
• determines the direction of the magnetic field around a currentcarrying wire and vice-versa
Gyromagnetic ratio (g)
•
•
related to the relative sensitive of the NMR signal
magnetic moment (m) is created along axis of the nuclear spin
m g p
g
2p m m

h I I
where:
p – angular momentum
g – gyromagnetic ratio (different value for each type of nucleus)
•
magnetic moment is quantized (m)
m = I, I-1, I-2, …, -I
for common nuclei of interest:
m = +½ & -½
Isotope
Net Spin
g / MHz T-1
Abundance / %
1H
1/2
42.58
99.98
2H
1
6.54
0.015
3H
1/2
45.41
0.0
31P
1/2
17.25
100.0
23Na
3/2
11.27
100.0
14N
1
3.08
99.63
15N
1/2
4.31
0.37
13C
1/2
10.71
1.108
19F
1/2
40.08
100.0
Magnetic alignment
= g h / 4p
Bo
In the absence of external field,
each nuclei is energetically degenerate
Add a strong external field (Bo) and the nuclear
magnetic moment: aligns with (low energy)
against (high-energy)
Spins Orientation in a Magnetic Field (Energy Levels)
•
•
Magnetic moment are no longer equivelent
Magnetic moments are oriented in 2I + 1 directions in magnetic field

Vector length is:

Angle (j) given by:

Energy given by:
 I ( I  1)
cos j 
E
m
I ( I  1)
mm
Bo
I
where,
Bo – magnetic Field
m – magnetic moment
h – Planck’s constant
For I = 1/2
Spins Orientation in a Magnetic Field (Energy Levels)
•
Magnetic moments are oriented in one of two directions in magnetic field (for I =1/2)
•
Difference in energy between the two states is given by:
DE = g h Bo / 2p
where:
Bo – external magnetic field
h – Planck’s constant
g – gyromagnetic ratio
Spins Orientation in a Magnetic Field (Energy Levels)
•
Transition from the low energy to high energy spin state occurs through an
absorption of a photon of radio-frequency (RF) energy
RF
Frequency of absorption:
n = g Bo / 2p
NMR Signal (sensitivity)
•
•
•
The applied magnetic field causes an energy difference between the
aligned (a) and unaligned (b) nuclei
NMR signal results from the transition of spins from the a to b state
Strength of the signal depends on the population difference between the a
and b spin states
b
DE = h n
Bo > 0
Bo = 0
•
Low energy gap
a
The population (N) difference can be determined from the Boltzmman
distribution and the energy separation between the a and b spin states:
Na / Nb = e DE / kT
NMR Signal (sensitivity)
Since:
DE = hn
and
n = g Bo / 2p
then:
Na / Nb = e DE / kT
Na/Nb = e(ghBo/2pkT)
The DE for 1H at 400 MHz (Bo = 9.39 T) is 6 x 10-5 Kcal / mol
Na / Nb  1.000060
Very Small !
~60 excess spins per
million in lower state
NMR Sensitivity
NMR signal (s) depends on: s % g4Bo2NB1g(u)/T
1) Number of Nuclei (N) (limited to field homogeneity and filling factor)
2) Gyromagnetic ratio (in practice g3)
3) Inversely to temperature (T)
4) External magnetic field (Bo2/3, in practice, homogeneity)
5)
B12 exciting field strength (RF pulse)
DE  g h Bo / 2p
Na / Nb = e DE /
kT
Increase energy gap -> Increase population difference -> Increase NMR signal
DE
≡
Bo ≡
g
NMR Sensitivity
Increase in Magnet
Strength is a Major Means
to Increase Sensitivity
NMR Sensitivity
But at a significant cost!
~$800,000
~$2,00,000
~$4,500,000
NMR Sensitivity
•
Relative sensitivity of 1H, 13C, 15N and other nuclei NMR spectra depend on

Gyromagnetic ratio (g)

Natural abundance of the isotope
g - Intrinsic property of nucleus can not be changed.
(gH/gC)3
1H
for
13C
is 64x (gH/gN)3 for
is ~ 64x as sensitive as
13C
15N
is 1000x
and 1000x as sensitive as
15N
!
Consider that the natural abundance of 13C is 1.1% and 15N is 0.37%
relative sensitivity increases to ~6,400x and ~2.7x105x !!
1H
NMR spectra of caffeine
8 scans ~12 secs
13C
NMR spectra of caffeine
8 scans ~12 secs
13C
NMR spectra of caffeine
10,000 scans ~4.2 hours
Theory of NMR
Classical Description
•
Spinning particle precesses around an applied magnetic field
A Spinning Gyroscope
in a Gravity Field
cos j 
m
I ( I  1)
Classical Description
•
Angular velocity of this motion is given by:
wo = gBo
where the frequency of precession or Larmor frequency is:
n = gBo/2p
Same as quantum mechanical description
Classical Description
•
Net Magnetization


Nuclei either align with or against external magnetic field along the z-axis.
Since more nuclei align with field, net magnetization (Mo, MZ) exists parallel
to external magnetic field.
Net Magnetization along +Z, since higher population aligned with Bo.
Magnetization in X,Y plane (MX,MY) averages to zero.


z
z
Mo
x
y
Bo
y
Bo
x
Classical Description
•
Observe NMR Signal

Need to perturb system from equilibrium.


Net magnetization (Mo) now precesses about Bo and B1




B1 field (radio frequency pulse) with gBo/2p frequency
MX and MY are non-zero
Mx and MY rotate at Larmor frequency
System absorbs energy with transitions between aligned and unaligned states
Precession about B1stops when B1 is turned off
Mz
RF pulse
B1 field perpendicular to B0
Mxy
Classical Description
•
Observe NMR Signal
Remember: a moving magnetic field perpendicular to a coil will induce a
current in the coil.
The induced current monitors the nuclear precession in the X,Y plane


X
y
Detect signal along X
RF pulse along Y
n = gBo/2p
Classical Description
•
Rotating Frame


To simplify the vector description, the X,Y axis rotates about the Z axis at
the Larmor frequency (X’,Y’)
B1 is stationary in the rotating frame
Mz
+
Mxy
Classical Description
•
NMR Pulse


Applying the B1 field for a specified duration (Pulse length or width)
Net Magnetization precesses about B1 a defined angle (90o, 180o, etc)
z
z
90o pulse
Mo
B1
w1
x
B1 off…
x
(or off-resonance)
y
w1 = gB1
y
Mxy w
1
Net Magnetization
z
z
Classic View:
- Nuclei either align with or
against external magnetic
field along the z-axis.
- Since more nuclei align with
field, net magnetization (Mo)
exists parallel to external
magnetic field
Mo
x
y
x
y
Bo
Bo
Quantum Description:
- Nuclei either populate low
energy (a, aligned with field)
or high energy (b, aligned
against field)
- Net population in a energy
level.
- Absorption of radiofrequency promotes nuclear
spins from a  b.
b
DE = h n
Bo > 0
a
Bo
Absorption of RF Energy or NMR RF Pulse
z
Classic View:
90o pulse
- Apply a radio-frequency (RF)
pulse a long the y-axis
- RF pulse viewed as a second
field (B1), that the net
magnetization (Mo) will
precess about with an
angular velocity of w1
--
z
Mo
B1
w1
x
B1 off…
x
(or off-resonance)
y
w1 = gB1
precession stops when B1
turned off
Mxy w
1
y
b
Quantum Description:
- enough RF energy has been
absorbed, such that the
population in a/b are now
equal
- No net magnetization along
the z-axis
DE = h n
a
Bo > 0
Please Note: A whole variety of pulse widths are possible, not quantized dealing
with bulk magnetization