NMR Spectroscopy by Venkatesh

8/3/2019 NMR Spectroscopy by Venkatesh

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NMR SPECTROSCOPY

VENKATESH GOUD

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Introduction to Spectroscopy2

Spectroscopy is the study of the interaction of matter with theelectromagnetic spectrum

1. Electromagnetic radiation displays the properties of bothparticles and waves

2. This packet of wave and particle properties is called a photon

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

3. The energy E component of a photon is proportional to thefrequency n

E = hn

The constant of proportionality is Planks constant, h


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Introduction to Spectroscopy3

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

5. Amplitude describes the wave height, or strength of the oscillation

6. Because the atomic particles in matter also exhibit wave and particleproperties (though opposite in how much) EM radiation can interact withmatter in two ways:

Collision particle-to-particle energy is lost as heat and

movement

Coupling the wave property of the radiation matches the waveproperty of the particle and couple to the next higher quantummechanical energy level

n = ___lc

E=hn = ___l

hc


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The Spectroscopic Process4

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1. Irradiation: Moleculeis bombarded withphotons of various

frequencies over therange desired

hn hn hn

5. Detection: Photons thatare reemitted and detected bythe spectrometer correspondto quantum mechanicalenergy levels of the molecule

Energy

2. Absorption:Moleculetakes on the quantum energyof a photon that matches theenergy of a transition and

becomes excited hn

rest state

rest state

excited state


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Basis of NMR Spectroscopy5

Nuclear Spin States

The sub-atomic particles within atomic nuclei possess a spinquantum number just like electrons

Just as when using Hunds rules to fill atomic orbitals withelectrons, nucleons must each have a unique set ofquantum numbers

The total spin quantum number of a nucleus is a physicalconstant, I

For each nucleus, the total number of spin states allowed isgiven by the equation:

2I+ 1


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6. Observe that for atoms with no net nuclear spin, there are zeroallowed spin states

7. Nuclear Magnetic Resonance can only occur where there areallowed spin states

8. Note that two nuclei, prevalent in organic compounds haveallowed nuclear spin states1H and 13C, while two others donot 12C and 16O

Spin Quantum Numbers of Common Nuclei

Element 1H 2H 12C 13C 14N 16O 17O 19F 31P 35Cl

Nuclear SpinQuantum Number

1 0 1 0 5/2 3/2

# of spin states 2 3 0 2 3 0 6 2 2 4


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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 ofprotons, the rotation of the nucleus will generate a magnetic

field along the axis of rotation

Thus, a nucleus has a magnetic moment, m, generated by itscharge and spin

A hydrogen atom with its lone proton making up the nucleus,can have two possible spin statesdegeneratein energy

m

H H

m

I = + I = -


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Which Elements or Moleculesare NMR Active?

Any atom or element with an odd number ofneutrons 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


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Nuclear Magnetic Resonance9

When a nucleiof spin +

encounters aphoton wheren = E/h, the two

couple

The nucleiflips its spin

state from +to and is nowopposed to B0

The nucleirelaxes with

the re-emissionof a photon andreturns to the+ spin state


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Nuclear Magnetic Resonance10

For the 1H nucleus (proton) this resonance condition occursat low energy (lots of noise) unless a very large magneticfield is applied

Early NMR spectrometers used a large permanent magnetwith a field of 1.4 Teslaprotons undergo resonance at 60MHz (1 MHz = 106 Hz)

Modern instruments use a large superconducting magnetour NMR operates at 9.4 T where proton resonance occurs at

400 MHz

In short, higher field gives cleaner spectra and allows longerand more detailed experiments to be performed


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Origin of the Chemical Shift11

Electronssurrounding the

nucleus are

opposite incharge to the

proton, thereforethey generatean opposing b0

Deshieding

Factors whichlower e- density

allow the nucleusto see more of

the B0 beingapplied

resonance occursat lowerenergy

Shielding

Factors which raisee- density reducethe amount of B0

the nucleus sees resonance

condition occurs athigherenergy


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The Proton (1H) NMR Spectrum12


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The 1H NMR Spectrum13

A reference compound is neededone that is inertand 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 resonancefor the protons on this compound is set to d 0.00

Si

CH3

H3C CH3CH3


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The 1H NMR Spectrum14

We need to consider four aspects of a 1Hspectrum:

a. Number of signals

b. Position of signals

c. Intensity of signals.

d. Spin-spin splitting of signals


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The Number of Signals15

The number of NMR signals equals the number ofdifferent types of protons in a compound

Protons in different environments give different

NMR signals

Equivalent protons give the same NMR signal


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To determine if two protons are chemically equivalent,substitute X for that each respective hydrogen in thecompound and compare the structures

If the two structures are fully superimposible (identical)the two hydrogens are chemically equivalent; if the twostructures are different the two hydrogens were notequivalent

A simple example: p-xyleneCH3

CH3

H

H

CH3

CH3

H

Z

CH3

CH3

Z

H

Same Compound


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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 ofSignals

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Remember:

Electrons

surrounding thenucleus areopposite in

charge to theproton, therefore

they generatean opposing b0

Deshieding

Factors whichlower e- density

allow the nucleusto see more of

the B0 beingapplied

resonance occursat lowerenergy

Shielding

Factors which raisee- density reducethe amount of B0

the nucleus sees resonance

condition occurs athigherenergy


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The less shielded the nucleus becomes, the more of theapplied magnetic field (B0) it feels

This deshielded nucleus experiences a higher magnetic fieldstrength, to it needs a higher frequency to achieve resonance

Higher frequency is to the left in an NMR spectrum, towardhigher chemical shiftso deshieldingshifts an absorptiondownfield

Downfield, deshielded Upfield, shielded


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There are three principle effects that contributeto local diamagnetic shielding:

a. Electronegativity

b. Hybridizationc. Proton acidity/exchange


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Electronegative groups comprise most organicfunctionalities:

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

-NHR -NR2 -NH3+ -C=O -NO2 -NO -

SO3H

-PO3H2 -SH -Ph -C=C and most others

In all cases, the inductive WD of electrons of thesegroups decreases the electron density in the C-Hcovalent bond proton is deshielded signal more

downfield of TMS


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Protons bound to carbons bearing electronwithdrawing groups are deshielded based on themagnitude of the withdrawing effect Paulingelectronegativity:

CH3F CH3O- CH3Cl CH3Br CH3I CH4 (CH3)4Si

PaulingElectronegativity

4.0 3.5 3.1 2.8 2.5 2.1 1.8

d of H 4.26 3.40 3.05 2.68 2.16 0.23 0.0


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3. The magnitude of the deshielding effect is cumulative:

As more chlorines are added d becomes larger

3. The magnitude of the deshielding effect is reduced bydistance, as the inductive model suggests

CH3Cl CH2Cl2 CHCl3

dof H 3.05 5.30 7.27

-CH2Br -CH2CH2Br -CH2CH2CH2Br

dofH 3.30 1.69 1.25

C S


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What we observe is slightly different:

Type of H Carbonhybridization

Name of H Chemical Shift, d

R-CH3, R2CH2, R3CH sp3 alkyl 0.8-1.7

C=C-CH3 sp3 allyl 1.6-2.6

CC-H sp acetylenic 2.0-3.0

C=C-H sp2 vinylic 4.6-5.7

Ar-H sp2 aromatic 6.5-8.5

O=C-H sp2 aldehydic 9.5-10.1

Chemists refer to this observation as magnetic anisotropy

Ch i l Shif P i i f


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Magnetic Anisotropy Aromatic Protonsa. In a magnetic field, the six electrons in benzene

circulate around the ring creating a ring current.

b. The magnetic field induced by these moving electronsreinforces the applied magnetic field in the vicinity of the

protons.c. The protons thus feel a stronger magnetic field and a

higher frequency is needed for resonance. Thus they aredeshielded and absorb downfield.

Ch i l Shif P i i f


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Similarly this effect operates in alkenes:

Ch i l Shif P i i f


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In alkynes there are two perpendicular sets of -electronsthe molecule orients with the fieldlengthwiseopposing B0shieldingthe terminal

H atom

Ch i l Shif P i i f


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Ch i l Shift P iti f


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Intensity of SignalsIntegration30

The area under an NMR signal is proportional to the number ofabsorbing protons

An NMR spectrometer automatically integrates the area under thepeaks, 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 thevalue of each integral in arbitrary units

The ratio of integrals to one another gives the ratio of absorbingprotons in a spectrum; note that this gives a ratio, and not theabsolute number, of absorbing protons


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Spin-Spin Splitting33

Consider the spectrum of ethyl alcohol:

Why does each resonance split into smaller

peaks?

HO

C

H2

CH3


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Structure Determination34


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13C NMR38

The lack of splitting in a 13C spectrum is aconsequence of the low natural abundance of13CA 13C NMR signal can also be split bynearby protons. This 1H-13C splitting is usually

eliminated from the spectrum by using aninstrumental technique that decouples theproton-carbon interactions, so that every peakin a 13C NMR spectrum appears as a singlet

The two features of a 13C NMR spectrum thatprovide the most structural information arethe number of signals observed and thechemical shifts of those signals


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13C NMR39


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13C NMR40

The number of signals in a 13C spectrum gives the number ofdifferent types of carbon atoms in a molecule.

Because 13C NMR signals are not split, the number of signals equalsthe number of lines in the 13C spectrum.

In contrast to the 1H NMR situation, peak intensity is not proportionalto the number of absorbing carbons, so 13C NMR signals are notintegrated.


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13C NMR41

In contrast to the small range of chemical shifts in 1H NMR (1-10 ppm usually), 13C NMR absorptions occur over a muchbroader range (0-220 ppm).

The chemical shifts of carbon atoms in 13C NMR depend onthe same effects as the chemical shifts of protons in 1H NMR.


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13C NMR42


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13C NMR43


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Principles of NMR

Measures nuclear magnetism or changes innuclear magnetism in a molecule

NMR spectroscopy measures the absorption oflight (radio waves) due to changes in nuclear spinorientation

NMR only occurs when a sample is in a strong

magnetic field Different nuclei absorb at different energies

(frequencies)


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INSTRUMENTATION

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Instrumentation for Spectroscopy


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Instrumentation for Spectroscopy

SourceWavelength

Selector

SampleDetector Data

Readout

Absorption Spectroscopy

Emission Spectroscopy

Source

WavelengthSelector

Sample

Detector DataReadout

WavelengthSelector

EXCITATION

EMISSION

Sources
http://www.tashika.co.jp/images/lamps.jpg


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Sources

Continuous Sources

Xenon Arc Lamp

250

600 nm Molecular Fluorescence

Hydrogen or Deuterium Lamp180380 nm

UV Molecular Absorbance

Tungsten/Halogen 2402500 nm

UV/Vis/NIRMolecular Absorbance

Tungsten3502200 nm

Vis/NIR

Nernst Glower400-20,000 nm

IR

molecular absorbance

Nichrome75020,000 nm

Globar120040,000 nm

Tunable Dye Lasers


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Different Types of NMR

Electron Spin Resonance (ESR)

1-10 GHz (frequency) used in analyzing freeradicals (unpaired electrons)

Magnetic Resonance Imaging (MRI)

50-300 MHz (frequency) for diagnostic imagingof soft tissues (water detection)

NMR Spectroscopy (MRS) 300-900 MHz (frequency) primarily used for

compound ID and characterization


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NMR in Everyday Life

Magnetic Resonance Imaging


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DRX-800 NMR spectrometer


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NMR Magnet


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An NMR Probe


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Magnet Legs

NMR Magnet Cross-Section


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A Modern NMR Instrument

Radio WaveTransceiver


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NMR Sample & Probe Coil


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Running an NMR Experiment

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; solid-phase 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!

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Running an NMR Experiment

Deuterated solvents are employedall 1H atomsreplaced with 2H which resonates at a differentfrequency

Most common: CDCl3 and D2O

Employed if necessary: CD2Cl2, DMSO-d6, toluene-d8,benzene-d

6, CD

3OD, acetone-d

6

Sample is contained in a high-tolerancethin glass tube (5 mm)

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Applications

Determination of exact structure of drugs anddrug metabolites - MOST POWERFUL METHODKNOWN

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 biofluidmixtures to quantify and identify metabolitechanges

Allows one to detect drug toxicity and evenlocalize toxicity (for preclinical trials) in a non-invasive way

Detection, identification and quantitation ofprimary and secondary drug metabolites


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Metabonomics

Alanine 0.84 mM Glutamine 0.60 mM

Arginine 0.70 mM Glycine 1.75 mM

Asparagine 0.72 mM Hippuric Acid 5.60 mM

Betaine 0.56 mM Hydroxybutyrate 1.12 mM

Citrate 1.68 mM Hydroxyproline 1.26 mM

Creatine 4.80 mM Isoleucine 1.05 mM

Creatinine 16.80 mM Phenylalanine 1.40 mMDimethylamine 1.80 mM Serine 0.84 mM

Dimethylglycine 3.50 mM Trimethylamine-N-Oxide 3.00 mM


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Other Applications

Clinical testing (detection of inborn errors ofmetabolism, 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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THANK YOU

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NMR Spectroscopy by Venkatesh

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