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NMR SPECTROSCOPY
VENKATESH GOUD

Introduction to Spectroscopy
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Spectroscopy is the study of the interaction of matter with the electromagnetic spectrum 1. Electromagnetic radiation displays the properties of both particles and waves
2.

This packet of wave and particle properties is called a photon

The term photon is implied to mean a small, massless particle that contains a small wave-packet of EM radiation/light
3.

The energy E component of a photon is proportional to the frequency n

E = hn
The constant of proportionality is Planks constant, h

Introduction to Spectroscopy
3 5.

Because the speed of light (c ) is constant, the frequency (n) (number of cycles of the wave per second) can complete in the same time, must be inversely proportional to how long the oscillation is, or wavelength (l):

n=
5.

c ___ l E = hn =

hc ___ l

Amplitude describes the wave height, or strength of the oscillation Because the atomic particles in matter also exhibit wave and particle properties (though opposite in how much) EM radiation can interact with matter in two ways: Collision particle-to-particle energy is lost as heat and movement

6.

Coupling the wave property of the radiation matches the wave

property of the particle and couple to the next higher quantum mechanical energy level

The Spectroscopic Process

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2. Absorption: Molecule takes on the quantum energy of a photon that matches the energy of a transition and becomes excited

excited state

hn

5. Detection: Photons that are reemitted and detected by the spectrometer correspond to quantum mechanical energy levels of the molecule

Energy

rest state 1. Irradiation: Molecule is bombarded with photons of various frequencies over the range desired

rest state

hn hn hn

Basis of NMR Spectroscopy

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Nuclear Spin States

The sub-atomic particles within atomic nuclei possess a spin quantum number just like electrons
Just as when using Hunds rules to fill atomic orbitals with electrons, nucleons must each have a unique set of quantum numbers The total spin quantum number of a nucleus is a physical constant, I For each nucleus, the total number of spin states allowed is given by the equation:
2I + 1

Basis of NMR Spectroscopy

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6.

Observe that for atoms with no net nuclear spin, there are zero allowed spin states Nuclear Magnetic Resonance can only occur where there are allowed spin states Note that two nuclei, prevalent in organic compounds have allowed nuclear spin states 1H and 13C, while two others do not 12C and 16O
1H 2H 12C 13C 14N 16O 17O 19F 31P 35Cl

7.

8.

Spin Quantum Numbers of Common Nuclei Element Nuclear Spin Quantum Number # of spin states

1 3

0 0

1 3

0 0

5/2 6

3/2 4

Basis of NMR Spectroscopy

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Nuclear Magnetic Moments

A nucleus contains protons, which each bear a +1 charge If the nucleus has a net nuclear spin, and an odd number of protons, the rotation of the nucleus will generate a magnetic field along the axis of rotation

I = +

I = -

Thus, a nucleus has a magnetic moment, m, generated by its charge and spin
A hydrogen atom with its lone proton making up the nucleus, can have two possible spin statesdegenerate in energy

Which Elements or Molecules are NMR Active?

Any atom or element with an odd number of neutrons and/or an odd number of protons Any molecule with NMR active atoms 1H - 1 proton, no neutrons, AW = 1 13C - 6 protons, 7 neutrons, AW =13 15N - 7 protons, 8 neutrons, AW = 15 19F = 9 protons, 10 neutrons, AW = 19

Nuclear Magnetic Resonance

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When a nuclei of spin + encounters a photon where n = E/h, the two couple

The nuclei flips its spin state from + to and is now opposed to B0

The nuclei relaxes with the re-emission of a photon and returns to the + spin state

Nuclear Magnetic Resonance

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For the 1H nucleus (proton) this resonance condition occurs at low energy (lots of noise) unless a very large magnetic field is applied Early NMR spectrometers used a large permanent magnet with a field of 1.4 Teslaprotons undergo resonance at 60 MHz (1 MHz = 106 Hz) Modern instruments use a large superconducting magnet our NMR operates at 9.4 T where proton resonance occurs at 400 MHz In short, higher field gives cleaner spectra and allows longer and more detailed experiments to be performed

Origin of the Chemical Shift

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Electrons surrounding the nucleus are opposite in charge to the proton, therefore they generate an opposing b0

Deshieding
Factors which lower e- density allow the nucleus to see more of the B0 being applied resonance occurs at lower energy

Shielding
Factors which raise e- density reduce the amount of B0 the nucleus sees resonance condition occurs at higher energy

The Proton (1H) NMR Spectrum

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The 1H NMR Spectrum

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A reference compound is neededone that is inert and does not interfere with other resonances Chemists chose a compound with a large number of highly shielded protonstetramethylsilane (TMS) No matter what spectrometer is used the resonance for the protons on this compound is set to d 0.00
CH3 Si H3C CH CH3 3

The 1H NMR Spectrum

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We need to consider four aspects of a 1H spectrum: a. Number of signals b. Position of signals c. Intensity of signals. d. Spin-spin splitting of signals

The Number of Signals

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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

The Number of Signals

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To determine if two protons are chemically equivalent, substitute X for that each respective hydrogen in the compound and compare the structures If the two structures are fully superimposible (identical) the two hydrogens are chemically equivalent; if the two structures are different the two hydrogens were not equivalent CH
3

A simple example: p-xylene

CH 3 H H CH 3 CH 3 CH 3

Same Compound
Z H CH 3

The Number of Signals

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Examples

Important: To determine equivalent protons in cycloalkanes and alkenes, always draw all bonds to show specific stereochemistry:

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Chemical Shift Position of Signals

Remember:

Electrons surrounding the nucleus are opposite in charge to the proton, therefore they generate an opposing b0

Deshieding
Factors which lower e- density allow the nucleus to see more of the B0 being applied resonance occurs at lower energy

Shielding
Factors which raise e- density reduce the amount of B0 the nucleus sees resonance condition occurs at higher energy

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Chemical Shift Position of Signals

The less shielded the nucleus becomes, the more of the applied magnetic field (B0) it feels This deshielded nucleus experiences a higher magnetic field strength, to it needs a higher frequency to achieve resonance

Higher frequency is to the left in an NMR spectrum, toward higher chemical shiftso deshielding shifts an absorption downfield
Downfield, deshielded Upfield, shielded

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Chemical Shift Position of Signals

There are three principle effects that contribute to local diamagnetic shielding:
a. b. c.

Electronegativity Hybridization Proton acidity/exchange

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Chemical Shift Position of Signals

Electronegative groups comprise most organic functionalities:

-F -Cl -Br -I -OH -OR -NH2

-NHR -NR2 SO3H -PO3H2 -SH

-NH3+

-C=O

-NO2

-NO

-Ph

-C=C

and most others

In all cases, the inductive WD of electrons of these groups decreases the electron density in the C-H covalent bond proton is deshielded signal more downfield of TMS

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Chemical Shift Position of Signals

Protons bound to carbons bearing electron withdrawing groups are deshielded based on the magnitude of the withdrawing effect Pauling electronegativity:
CH3F Pauling Electronegativity d of H 4.0 4.26 CH3O3.5 3.40 CH3Cl 3.1 3.05 CH3Br 2.8 2.68 CH3I 2.5 2.16 CH4 2.1 0.23 (CH3)4Si 1.8 0.0

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Chemical Shift Position of Signals

3.

The magnitude of the deshielding effect is cumulative:

CH3Cl d of H 3.05 CH2Cl2 5.30 CHCl3 7.27

As more chlorines are added d becomes larger

3.

The magnitude of the deshielding effect is reduced by distance, as the inductive model suggests
-CH2Br -CH2CH2Br -CH2CH2CH2Br

d of H

3.30

1.69

1.25

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Chemical Shift Position of Signals

What we observe is slightly different:

Type of H R-CH3, R2CH2, R3CH C=C-CH3 CC-H C=C-H Carbon hybridization sp3 sp3 sp sp2 Name of H alkyl allyl acetylenic vinylic Chemical Shift, d 0.8-1.7 1.6-2.6 2.0-3.0 4.6-5.7

Ar-H
O=C-H

sp2
sp2

aromatic
aldehydic

6.5-8.5
9.5-10.1

Chemists refer to this observation as magnetic anisotropy

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Chemical Shift Position of Signals

Magnetic Anisotropy Aromatic Protons

a. b.

c.

In a magnetic field, the six electrons in benzene circulate around the ring creating a ring current. The magnetic field induced by these moving electrons reinforces the applied magnetic field in the vicinity of the protons. The protons thus feel a stronger magnetic field and a higher frequency is needed for resonance. Thus they are deshielded and absorb downfield.

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Chemical Shift Position of Signals

Similarly this effect operates in alkenes:

Chemical Shift Position of Signals

In alkynes there are two perpendicular sets of electronsthe molecule orients with the field lengthwiseopposing B0 shielding the terminal H atom

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Chemical Shift Position of Signals

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Chemical Shift Position of Signals

Intensity of SignalsIntegration
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The area under an NMR signal is proportional to the number of absorbing protons An NMR spectrometer automatically integrates the area under the peaks, and prints out a stepped curve (integral) on the spectrum The height of each step is proportional to the area under the peak, which in turn is proportional to the number of absorbing protons Modern NMR spectrometers automatically calculate and plot the value of each integral in arbitrary units The ratio of integrals to one another gives the ratio of absorbing protons in a spectrum; note that this gives a ratio, and not the absolute number, of absorbing protons

Intensity of SignalsIntegration
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Intensity of SignalsIntegration
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Spin-Spin Splitting
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Consider the spectrum of ethyl alcohol: Why does each resonance split into smaller peaks?
HO C H2 CH3

Structure Determination
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Structure Determination
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Structure Determination
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Structure Determination
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13C
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NMR

The lack of splitting in a 13C spectrum is a consequence of the low natural abundance of 13CA 13C NMR signal can also be split by nearby protons. This 1H-13C splitting is usually eliminated from the spectrum by using an instrumental technique that decouples the proton-carbon interactions, so that every peak in a 13C NMR spectrum appears as a singlet The two features of a 13C NMR spectrum that provide the most structural information are the number of signals observed and the chemical shifts of those signals

13C
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NMR

13C
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NMR

The number of signals in a 13C spectrum gives the number of different types of carbon atoms in a molecule. Because 13C NMR signals are not split, the number of signals equals the number of lines in the 13C spectrum. In contrast to the 1H NMR situation, peak intensity is not proportional to the number of absorbing carbons, so 13C NMR signals are not integrated.

13C
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NMR

In contrast to the small range of chemical shifts in 1H NMR (110 ppm usually), 13C NMR absorptions occur over a much broader range (0-220 ppm). The chemical shifts of carbon atoms in 13C NMR depend on the same effects as the chemical shifts of protons in 1H NMR.

13C
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NMR

13C
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NMR

Principles of NMR

Measures nuclear magnetism or changes in nuclear magnetism in a molecule NMR spectroscopy measures the absorption of light (radio waves) due to changes in nuclear spin orientation NMR only occurs when a sample is in a strong magnetic field Different nuclei absorb at different energies (frequencies)

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INSTRUMENTATION

Instrumentation for Spectroscopy

Absorption Spectroscopy

Source Wavelength Selector

Sample

Detector

Data Readout

Emission Spectroscopy
Sample
EMISSION

Wavelength Selector

EXCITATION

Wavelength Selector

Detector

Data Readout

Source

Sources

Continuous Sources

Xenon Arc Lamp 250 600 nm

Molecular Fluorescence UV Molecular Absorbance UV/Vis/NIR Molecular Absorbance Vis/NIR IR molecular absorbance

Hydrogen or Deuterium Lamp 180 380 nm

Tungsten/Halogen 240 2500 nm

Tungsten 350 2200 nm

Nernst Glower 400-20,000 nm

Nichrome 750 20,000 nm Globar 1200 40,000 nm Tunable Dye Lasers

Different Types of NMR

Electron Spin Resonance (ESR)

1-10

GHz (frequency) used in analyzing free radicals (unpaired electrons) MHz (frequency) for diagnostic imaging of soft tissues (water detection) MHz (frequency) primarily used for compound ID and characterization

Magnetic Resonance Imaging (MRI)

50-300

NMR Spectroscopy (MRS)

300-900

NMR in Everyday Life

Magnetic Resonance Imaging

DRX-800 NMR spectrometer

NMR Magnet

An NMR Probe

NMR Magnet Cross-Section

Magnet Legs

A Modern NMR Instrument

Radio Wave Transceiver

NMR Sample & Probe Coil

Running an NMR Experiment

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Sample sizes for a typical high-field NMR (300-600 MHz):

1-10 mg for 1H NMR 10-50 mg for 13C NMR

Solution phase NMR experiments are much simpler to run; solidphase NMR requires considerable effort Sample is dissolved in ~1 mL of a solvent that has no 1H hydrogens

Otherwise the spectrum would be 99.5% of solvent, 0.5% sample!

Running an NMR Experiment

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Deuterated solvents are employedall 1H atoms replaced with 2H which resonates at a different frequency Most common: CDCl3 and D2O Employed if necessary: CD2Cl2, DMSO-d6, toluene-d8, benzene-d6, CD3OD, acetone-d6 Sample is contained in a high-tolerance thin glass tube (5 mm)

Applications

Determination of exact structure of drugs and drug metabolites - MOST POWERFUL METHOD KNOWN Detection/quantitation of impurities Detection of enantiomers (shift reagents) High throughput drug screening Analysis/deconvolution of liquid mixtures Water content measurement

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Metabonomics

Analysis of blood, urine and other biofluid mixtures to quantify and identify metabolite changes Allows one to detect drug toxicity and even localize toxicity (for preclinical trials) in a noninvasive way Detection, identification and quantitation of primary and secondary drug metabolites

Metabonomics
Alanine 0.84 mM Arginine 0.70 mM Asparagine 0.72 mM Betaine 0.56 mM Citrate 1.68 mM Creatine 4.80 mM Creatinine 16.80 mM Dimethylamine 1.80 mM Dimethylglycine 3.50 mM Glutamine Glycine Hippuric Acid Hydroxybutyrate Hydroxyproline Isoleucine Phenylalanine Serine Trimethylamine-N-Oxide 0.60 1.75 5.60 1.12 1.26 1.05 1.40 0.84 3.00 mM mM mM mM mM mM mM mM mM

Other Applications

Clinical testing (detection of inborn errors of metabolism, cancer, diabetes, organic solvent poisoning, drugs of abuse, etc. etc.) Cholesterol and lipoprotein testing Chemical Shift Imaging (MRI + MRS) Pharmaceutical Biotechnology (proteins, protein drugs, SAR by NMR)

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