SPECIAL TOPICS IN ANALYTICAL CHEMISTRY Introduction to NMR CHEM 921, Fall 2005

SPECIAL TOPICS IN ANALYTICAL CHEMISTRYIntroduction to NMRCHEM 921, Fall 2005

MWF 11:30-12:20pm, Rm 548 Hamilton Hall

COURSE OUTLINE

Instructor: Dr. Robert Powers Office Labs

Address: 722 HaH 720 HaHPhone: 472-3039 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: 50 pts (Fri., Sept. 9th)Exam 1: 100 pts (Fri., Sept 23rd)Exam 2: 100 pts (Wed., Nov. 2nd)Structure Problems (5) 150 pts (as assigned)Total: 400 pts

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 #4 Fri. Oct. 21st


• Grading (30 points/structure; 150 total points)

Lecture TopicsTopic Chapters in NMR & Chemistry

I. BASIC NMR TheoryI. Introduction to NMR Theory 1

a. Quantum and classical descriptionII. Obtaining an NMR Spectra 5

a. Data acquisition and processingb. Instrumentation

III. Chemical Shifts () 2a. Select examples of chemical shift trendsb. Predicting chemical shifts

IV. Coupling Constants (J) 3a. Simulation of second-order spin systems

II. One and Two Dimensional NMRI. NMR Pulses 6II. 1D NMR 8

a. NOE, J modulation, INEPT, DEPT, INADEQUATEIII. 2D NMR 9

a. Theoryb. COSY,TOCSY,NOESY,HMQC,HMBC

IV. Examples of Spectral Interpretation 8III. NMR Dynamics

• Relaxation 4I. T1,T2,Dipole-Dipole,CSA,Quadrupolar

I. Exchange 7a. NMR time scale

IV. Solid State NMR (as time permits) 11

>>> “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 Suggested NMR ReferencesSome Suggested NMR References

Some NMR Web SitesSome NMR Web Sites

The Basics of NMR by J.P. Hornak The Basics of NMR by J.P. Hornak Hypertext based NMR courseHypertext based NMR course http://www.cis.rit.edu/htbooks/nmr/nmr-main.htm

Integrated Spectral Data Base System for Organic Compoundshttp://www.aist.go.jp/RIODB/SDBS/menu-e.html Educational NMR Software All kinds of NMR softwarehttp://www.york.ac.uk/depts/chem/services/nmr/edusoft.html

NMR Knowledge Base A lot of useful NMR linkshttp://www.spectroscopynow.com/

NMR Information Server News, Links, Conferences, Jobshttp://www.spincore.com/nmrinfo/

Technical Tidbits Useful source for the art of shimminghttp://www.acornnmr.com/nmr_topics.htm

BMRB (BioMagResBank) Database of NMR resonance assignmentshttp://www.bmrb.wisc.edu/

http://www.cis.rit.edu/htbooks/nmr/nmr-main.htm

http://www.aist.go.jp/RIODB/SDBS/menu-e.html

http://www.york.ac.uk/depts/chem/services/nmr/edusoft.html

http://www.spectroscopynow.com/

http://www.spincore.com/nmrinfo/

http://www.acornnmr.com/nmr_topics.htm

http://www.bmrb.wisc.edu/



1937 Rabi predicts and observes nuclear magnetic resonance1946 Bloch, Purcell first nuclear magnetic resonance of bulk sample1953 Overhauser NOE (nuclear Overhauser effect)1966 Ernst, Anderson Fourier transform NMR1975 Jeener, Ernst 2D NMR1985 Wüthrich first solution structure of a small protein (BPTI)

from NOE derived distance restraints1987 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 relaxationTROSY (molecular weight > 100 kDa)

Nobel prizes1944 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 HistoryNMR History

NMR HistoryNMR History First NMR Spectra on Water

Bloch, F.; Hansen, W. W.; Packard, M. Bloch, F.; Hansen, W. W.; Packard, M. The nuclear induction experiment.The nuclear induction experiment. Physical Review (1946), 70 474-85. Physical Review (1946), 70 474-85.

11H NMR spectra of waterH NMR spectra of water

NMR HistoryNMR History First Observation of the Chemical Shift

11H NMR spectra ethanolH NMR spectra ethanol

Modern ethanol spectra Modern ethanol spectra

Arnold, J.T., S.S. Dharmatti, and M.E. Packard, J. Chem. Phys., 1951. Arnold, J.T., S.S. Dharmatti, and M.E. Packard, J. Chem. Phys., 1951. 1919: p. 507. : p. 507.

Nuclear Magnetic ResonanceNuclear 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 One of the MOSTMOST Routinely used Analytical Techniques Routinely used Analytical Techniques

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

NMR Structure of MMP-13 complexed to a ligand

O

O

O

O

OH

OO

O

HO

NH

OH

OO

O

O

Taxol (natural product)

2-phenyl-1,3-dioxep-5-ene2-phenyl-1,3-dioxep-5-ene

1313C NMR spectraC NMR spectra

11H NMR spectraH NMR spectra

Each NMR Observable Nuclei Yields a Peak in the SpectraEach NMR Observable Nuclei Yields a Peak in the Spectra““fingerprint” of the structurefingerprint” of the structure

Information in a NMR SpectraInformation in a NMR Spectra

1) Energy E = h

h is Planck constant is NMR resonance frequency 10-10 10-8 10-6 10-4 10-2 100 102

wavelength (cm)

-rays x-rays UV VIS IR -wave radio

ObservableObservable NameName QuantitativeQuantitative InformationInformation

Peak position Chemical shifts () (ppm) = obs –ref/ref (Hz) chemical (electronic)

environment of nucleus

Peak Splitting Coupling Constant (J) Hz peak separation neighboring nuclei (intensity ratios) (torsion angles)

Peak Intensity Integral unitless (ratio) nuclear count (ratio) relative height of integral curve T1 dependent

Peak Shape Line width = 1/T2 molecular motion peak half-height chemical exchange

uncertainty principaluncertainty in

energy

A Basic Concept in ElectroMagnetic TheoryA 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

For a single loop of wire, the magnetic field, B through the center of the loop is:

o – permeability of free space (4 x 10-7 T · m / A) R – radius of the wire loopI – current


RIB o2

Faraday’s Law of InductionFaraday’s Law of Induction• If the magnetic flux (B) 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:

dt

d B


Simple AC generator

Lenz’s LawLenz’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

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

1) quantized, spin quantum number I

2) 2I + 1 states: I, I-1, I-2, …, -II=1/2: -1/2, 1/2

3) identical energies in absence of external magnetic field

l

Theory of NMRTheory of NMR

)1( II

NMR Periodic TableNMR Periodic Table

NMR “active” Nuclear Spin (I) = ½: 1H, 13C, 15N, 19F, 31P biological and chemical relevanceOdd atomic mass

I = +½ & -½

NMR “inactive” Nuclear Spin (I) = 0:12C, 16OEven atomic mass & number

Quadrupole Nuclei Nuclear Spin (I) > ½:14N, 2H, 10B

Even atomic mass & odd numberI = +1, 0 & -1

Magnetic Moment (Magnetic Moment ())• spinning charged nucleus creates a magnetic field

Similar to magnetic field created by electric current flowing in a coil

Magnetic moment

““Right Hand Rule”Right Hand Rule”• determines the direction of the magnetic field around a current-carrying wire and vice-versa

Gyromagnetic ratio (Gyromagnetic ratio ())• related to the relative sensitive of the NMR signal• magnetic moment () is created along axis of the nuclear spin

where: p – angular momentum – 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

IIh

2p

Isotope Net Spin / 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

Bo

= h / 4

Magnetic alignmentMagnetic alignment

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)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:Vector length is:

Angle (Angle () given by:) given by:

Energy given by:Energy given by:

)1( II

)1(cos

II

m

oBI

mE

where,where,BBoo – magnetic Field – magnetic Field

– – magnetic momentmagnetic momenthh – Planck’s constant – Planck’s constant

For I = 1/2

Spins Orientation in a Magnetic Field (Energy Levels)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:

E = h Bo / 2

where:Bo – external magnetic fieldh – Planck’s constant – gyromagnetic ratio

Frequency of absorption: = Bo / 2

Spins Orientation in a Magnetic Field (Energy Levels)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

NMR Signal (sensitivity)NMR Signal (sensitivity)• The applied magnetic field causes an energy difference between the

aligned () and unaligned () nuclei• NMR signal results from the transition of spins from the to state• Strength of the signal depends on the population difference between the

and spin states

• The population (N) difference can be determined from the Boltzmman distribution and the energy separation between the and spin states:

N / N = e E / kT

Bo = 0

Bo > 0 E = h

Low energy gap

NMR Signal (sensitivity)NMR Signal (sensitivity)

Since:

E = hE = hand

= Bo / 2then:

The E for 1H at 400 MHz (Bo = 9.39 T) is 6 x 10-5 Kcal / mol

Very Small !Very Small !~60 excess spins ~60 excess spins per million in lower per million in lower statestate

N/N = e(hBo/2kT)N / N = e E / kT

NMR SensitivityNMR Sensitivity

EhBo /2

NMR signal (s) depends on:

1) Number of Nuclei (N) (limited to field homogeneity and filling factor)

2) Gyromagnetic ratio (in practice 3)

3) Inversely to temperature (T)

4) External magnetic field (Bo2/3, in practice, homogeneity)

5) B12 exciting field strength (RF pulse)

N / N = e E /

kT

Increase energy gap -> Increase population difference -> Increase NMR signalIncrease energy gap -> Increase population difference -> Increase NMR signal

E ≡ Bo≡

s 44BBoo22NBNB11g(g()/T)/T


Increase in Magnet Strength is a Major Means to Increase Sensitivity


But at a significant cost!

~$800,000 ~$2,00,000 ~$4,500,000

- Intrinsic property of nucleus can not be changed.

C)3 for 13C is 64xN)3

for 15N is 1000x

1H is ~ 64x as sensitive as 13C 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 !!


• Relative sensitivity of 1H, 13C, 15N and other nuclei NMR spectra depend on Gyromagnetic ratio (Gyromagnetic ratio ()) Natural abundance of the isotope Natural abundance of the isotope

1H NMR spectra of caffeine8 scans ~12 secs

13C NMR spectra of caffeine8 scans ~12 secs

13C NMR spectra of caffeine10,000 scans ~4.2 hours

Classical Description

Theory of NMRTheory of NMR

• Spinning particle precesses around an applied magnetic field

A Spinning Gyroscopein a Gravity Field

)1(cos

II

m


• Angular velocity of this motion is given by:

o = Bo

where the frequency of precession or LarmorLarmor frequency is:

= Bo/2

Same as quantum mechanical 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.

Mo

z

x

yy

x

z

Bo Bo

RF pulse

B1 field perpendicular to B0Mxy

Mz


• Observe NMR Signal Need to perturb system from equilibrium.

B1 field (radio frequency pulse) with Bo/2frequency Net magnetization (Mo) now precesses about Bo and B1

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

= Bo/2

RF pulse along Y

Detect signal along X

X

y


• 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


• 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

Mxy

Mz

+


• 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)

B1 off…

(or off-resonance)

Mo

z

x

B1

z

x

Mxy

y y1

1

1 = B1

90o pulse

Net Magnetization

Mo

z

x

yy

x

z

Bo Bo

Bo > 0 E = h

Bo

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

Quantum Description:- Nuclei either populate low energy (, aligned with field) or high energy (, aligned against field)

- Net population in energy level.

- Absorption of radio- frequency promotes nuclear spins from .

Absorption of RF Energy or NMR RF Pulse

Classic View:- 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 1

-- precession stops when B1 turned off

Quantum Description:- enough RF energy has been absorbed, such that the population in / are now equal

- No net magnetization along the z-axis

B1 off…

(or off-resonance)

Mo

z

x

B1

z

x

Mxy

y y1

1

1 = B1

90o pulse

Bo > 0

E = h

Please Note: A whole variety of pulse widths are possible, not quantized dealing with bulk magnetization

Documents

SPECIAL TOPICS IN ANALYTICAL CHEMISTRY Introduction to NMR CHEM 921, Fall 2005