Chapter10 140330084647-phpapp02

Chapter 10: NuclearChapter 10: Nuclear
Magnetic Resonance (NMR)Magnetic Resonance (NMR)
SpectroscopySpectroscopy

What is spectroscopy?What is spectroscopy?
Molecular response to radiative stimulus isMolecular response to radiative stimulus is
quantizedquantized (“geared”). ((“geared”). (Molecule = nuclei + electronsMolecule = nuclei + electrons).).
Excitation:Excitation:
1. Electronic (UV-visible spectra)1. Electronic (UV-visible spectra)
2. Vibrational (IR)2. Vibrational (IR)
3. Rotation (microwave)3. Rotation (microwave)
4. Nuclear spin orientation in magnet (NMR)4. Nuclear spin orientation in magnet (NMR)
SpectrometerSpectrometer scansscans νν to findto find ΔΔEE :: The spectrumThe spectrum
ΔΔEE == hhνν νν = c/= c/λλ

Excitation causesExcitation causes
an “an “absorptionabsorption”, a”, a
peakpeak in a graphin a graph

A GeneralA General
SpectrometerSpectrometer

What is ∆What is ∆EE in NMR? Nucleiin NMR? Nuclei
behave as tinybehave as tiny magnetsmagnets (random(random
orientation).orientation).
In aIn a magnetic field Hmagnetic field H00, they, they
organizeorganize with (with (αα, lower energy), lower energy) oror
againstagainst ((ββ, higher energy), higher energy) the field.the field.
∆∆EE = h= hνν00 resonance frequency:resonance frequency:
The frequency thatThe frequency that matchesmatches
exactly theexactly the energy differenceenergy difference..

Protons as Tiny Magnets Line UpProtons as Tiny Magnets Line Up
With and Against an ExternalWith and Against an External
Magnetic FiledMagnetic Filed
RatioRatio αα::ββ ~ 1:1~ 1:1

Absorption of Light, Spin Flip,Absorption of Light, Spin Flip,
and Resonanceand Resonance ⇒⇒ Spectral LineSpectral Line

νν00 isis proportionalproportional toto HH00.. ((νν00
isis specificspecific forfor element/isotopeelement/isotope))
ForFor 11
H: atH: at HH00 = 21,150 gauss,= 21,150 gauss, νν00 = 90 MHz.= 90 MHz.
∆∆EE (300 MHz) ~ 10(300 MHz) ~ 10-5-5
kcal molkcal mol-1-1
..
NNαα//NNββ = 1.000004= 1.000004
NMR “NMR “activeactive” nuclei:” nuclei: 11
HH andand 1313
CC ((notnot 1212
CC))
AtAt HH00 = 70500 gauss,= 70500 gauss, νν00 = 300 MHz.= 300 MHz.
HH00 (earth): 0.7 gauss!(earth): 0.7 gauss!

A Hypothetical NMRA Hypothetical NMR
Spectrum: Active ElementsSpectrum: Active Elements
Absorb at DifferingAbsorb at Differing
FrequenciesFrequencies

The NMR SpectrometerThe NMR Spectrometer
Solvents: CDClSolvents: CDCl33, CD, CD22ClCl22, THF-, THF-dd88, etc., etc.

High Resolution NMR!High Resolution NMR!

Why are there two peaks?Why are there two peaks? The Chemical ShiftThe Chemical Shift

CausesCauses shieldingshielding, i.e.,, i.e.,
Peak moves to the rightPeak moves to the right
Electrons in Vicinity of Nucleus AffectElectrons in Vicinity of Nucleus Affect νν

Consider HConsider H++
: No: No ee,, no shieldingno shielding, peak, peak
furthest to thefurthest to the leftleft..
But:But:
When we add anWhen we add an e-withdrawinge-withdrawing group:group:
e.g.,e.g., CHCH33 ClCl,, causescauses deshieldingdeshielding (to(to leftleft).).
Chemical shift provides a finely tuned pictureChemical shift provides a finely tuned picture
ofof electronic environmentelectronic environment around each H.around each H.
CC HHHH
HH
HH
++ --
ElectronsElectrons causecause shieldingshielding::
Peak movesPeak moves upfieldupfield (to(to rightright).).

If we substitute withIf we substitute with e-negativee-negative groups, shielding ofgroups, shielding of
observed nucleus isobserved nucleus is diminisheddiminished; or nucleus is; or nucleus is
““deshieldeddeshielded” (relatively).” (relatively).

One e-poorOne e-poor
neighborneighbor
Two e-poorTwo e-poor
neighborsneighbors

The Chemical ShiftThe Chemical Shift δδ
The position of peak relative to anThe position of peak relative to an
internal standard. Most common isinternal standard. Most common is
Tetramethylsilane: (CHTetramethylsilane: (CH33))44SiSi oror “TMS”“TMS”,,
thethe “zero”“zero” point in the spectrum.point in the spectrum.
==
νν (from TMS)(from TMS) HzHz
RF (e.g., 300 MHz)RF (e.g., 300 MHz)
ppmppm,,
isis independentindependent of machine (90, 300 MHz, etc.)of machine (90, 300 MHz, etc.)
δδ

Distance from
TMS in Hz/300
MHz in ppm
266 Hz
0.89 ppm
541 Hz
1.80 ppm
978 Hz
3.26 ppm

Application ofApplication of δδ
CHCH33 CHCH33 CHCH33CHCH22BrBr
BrBr22
hhυυ
+ CH+ CH33CHBrCHBr22 + BrCH+ BrCH22CHCH22BrBr
EPM of 1-bromopropaneEPM of 1-bromopropane
2 signals2 signals
2 signals2 signals
1 signal1 signal
Most deshieldedMost deshielded
of allof all

1-21-2
2+2+
3-43-4
VariableVariable

ChemicallyChemically equivalentequivalent HsHs:: samesame δδs.s. UseUse
symmetry operations: rotation, mirror planes.symmetry operations: rotation, mirror planes.

““Fast” processes, such as methylFast” processes, such as methyl
rotation, equilibrate Hs:rotation, equilibrate Hs:
For calibration: A BFor calibration: A B
EEaa
kk
EEaa ~ 20 kcal mol~ 20 kcal mol-1-1
,, kk ~ 10~ 10-2-2
secsec-1-1
, t, t1/21/2 = 1 min= 1 min
EEaa ~ 25 kcal mol~ 25 kcal mol-1-1
,, kk ~ 10~ 10-6-6
secsec-1-1
, t, t1/21/2 = 66 h= 66 h
NMR time scaleNMR time scale << 1 sec1 sec..

Recall:Recall: EEaa = 10.8 kcal mol= 10.8 kcal mol-1-1
..
ToTo “freeze” ring flip“freeze” ring flip on the NMR time scale:on the NMR time scale:
we need towe need to cool to -90ºCcool to -90ºC (Jensen)(Jensen)

IntegrationIntegration
Area under peakArea under peak == relative # ofrelative # of
associated Hs. Computer-automatedassociated Hs. Computer-automated..
e.g. (Ce.g. (CHH33))33CCCCHH22OOHH 99::22::11
CCHH33OCOCHH22CHCH22OCOCHH33 33::22
Helps in peak assignments.Helps in peak assignments.

Spin-Spin SplittingSpin-Spin Splitting

(N+1) Rule(N+1) Rule
NN equivalentequivalent adjacent hydrogensadjacent hydrogens
(i.e. on neighboring carbons) give(i.e. on neighboring carbons) give
rise to (rise to (N+1N+1) peaks.) peaks.
e.g., doublet (d), triplet (t),e.g., doublet (d), triplet (t),
quartet (q), quintet (quin), etc.quartet (q), quintet (quin), etc.

Why ?Why ?
HHbbαα strengthensstrengthens HH00 around Haround Haa :: deshieldingdeshielding
HHbbββ weakensweakens HH00 around Haround Haa :: shieldingshielding
Result:Result: two linestwo lines (1:1 ratio,(1:1 ratio, dd) instead of one.) instead of one.
Distance between them in Hz is theDistance between them in Hz is the
coupling constantcoupling constant JJ..
Cl C C O CHCl C C O CH22 CHCH33
HHaa
OCHOCH22CHCH33ClCl
HHbb
dd dd
qq tt
HHaa “sees” two types of H“sees” two types of Hbb neighborsneighbors
(through bond): H(through bond): Hbbαα and Hand Hbbββ
In a magnetic field, allIn a magnetic field, all
HHss exist as Hexist as Hαα:H:Hββ ~ 1:1.~ 1:1.

Same valueSame value
HHaa and Hand Hbb are said toare said to “split” each other“split” each other with awith a JJ of 7 Hz.of 7 Hz.

JJ isis field independent:field independent: Same at 90,Same at 90,
300, etc. MHz300, etc. MHz
Range: 1-18 Hz.Range: 1-18 Hz.
ForFor moremore than onethan one neighbor : H “sees”neighbor : H “sees”
allall αα,, ββ combinations of neighbors. Thus,combinations of neighbors. Thus,
twotwo neighboring Hs:neighboring Hs:
αααα,, αβαβ,, βαβα ,, ββββ: 1:: 1:22:1:1 triplettriplet
αααααα,, ααβααβ,, αβααβα,, βααβαα ,, αββαββ,, βαββαβ,, ββαββα ,, ββββββ
1:1:33::33:1:1 quartetquartet
ThreeThree neighboring Hs:neighboring Hs:

Summary PointsSummary Points
1.1. Equivalent protonsEquivalent protons showshow no splittingno splitting..
2.2. JJ isis independentindependent of Hof H00..
3. Nonadjacent Hs3. Nonadjacent Hs JJ ~ 0~ 0..
4.4. (N+1) Rule(N+1) Rule. Ratio from Pascal’s triangle.. Ratio from Pascal’s triangle.
5.5. Splitting is mutualSplitting is mutual. If there is one split peak,. If there is one split peak,
there has to be (at least) one other.there has to be (at least) one other.
CC XX CC
HHHH

(CH(CH33))33CHCH
decetdecet
tt
dd
tridecettridecet
All –CHAll –CH22- hydrogens are- hydrogens are
equivalent:equivalent:
ss
Four mirror planesFour mirror planes
tt
quintetquintet
OOOO
H

A 3.427 B 1.679 ppm
http://www.aist.go.jp/RIODB/SDBS/cgi-bin/cre_index.cgihttp://www.aist.go.jp/RIODB/SDBS/cgi-bin/cre_index.cgi
CHCH33CHCH33 CHCH33CHCH22Br + CHBr + CH33CHBrCHBr22 + BrCH+ BrCH22CHCH22BrBr
BrBr22
hh
tt qq dd qq singletsinglet

Problem:Problem:
3 H3 H
2 H2 H
1 H1 H
4 H4 H
3 H3 H
3 H3 H
2 H2 H
1 H1 H
1 H1 H
The two butanols:The two butanols:
CHCH33CHCH22CHCH22CHCH22OHOH
CHCH33CHCH22CHCHCHCH33
Which is which?Which is which?
OHOH

Complex SplittingComplex Splitting
PatternsPatterns
[N+1] rule holds strictly only when:[N+1] rule holds strictly only when:
1.1. JJs to all neighbors equal, ands to all neighbors equal, and
2.2. ∆∆νν >>>> JJ :: first order patternsfirst order patterns..
3.3. Otherwise complex signals:Otherwise complex signals: non-first ordernon-first order
multiplets.multiplets.
δδ (H(Heqeq) >) > δδ (H(Haxax))
11
11
44
22
33

Multiplets are typical of alkyl chains: allMultiplets are typical of alkyl chains: all δδs similar.s similar.
e.g.,e.g.,
One solution: stronger magnetOne solution: stronger magnet higher field spectrahigher field spectra
For example, what might be non-first-order
at 90 MHz, may resolve into a first-order
pattern at 300 or 500 MHz.
(Recall(Recall νν ~ H~ H00 ), “spreads out” spectrum,), “spreads out” spectrum,
butbut JJs stay same (s stay same (field independentfield independent).).

Ribonuclease A, 40 MHz
Ribonuclease ARibonuclease A

““Sequential” [N+1] RuleSequential” [N+1] Rule
11stst
Order patterns can be analyzedOrder patterns can be analyzed
for neighbors with differingfor neighbors with differing JJs.s.
JJs should differ fors should differ for non-equivalentnon-equivalent
neighborsneighbors..
CCHH33 CC CC ClCl
ClClClCl
HHbb HHaa
cc
JJaabb = 3.6 Hz= 3.6 Hz
JJbbcc = 6.8 Hz= 6.8 Hz
Apply split sequentiallyApply split sequentially

CCHH33 CC CC ClCl
ClClClCl
HHbb HHaa
cc
JJaabb = 3.6 Hz= 3.6 Hz
JJbbcc = 6.8 Hz= 6.8 Hz

CHCH33—C—CHH22—CH—CH22—Br—Br
((JJ22))
tt
??((JJ11))tt
JJ11 >> JJ22 ::
JJ11 << JJ22 ::
JJ11
JJ22
JJ22
JJ11
qtqt
oror
tqtq
Quartet of triplets or triplet ofQuartet of triplets or triplet of
quartets or….. ?quartets or….. ?

JJabab == JJbcbc  N + 1 RuleN + 1 Rule
IfIf JJs accidentally the same:s accidentally the same: [N+1] rule[N+1] rule
SextetSextet

Stereocenters and MethyleneStereocenters and Methylene
GroupsGroups

H Bonded/Acidic Protons:H Bonded/Acidic Protons:
OH, SH, N HOH, SH, N H
Recall CRecall CHH22 OO HH
Why?Why? FastFast HH++
exchangeexchange
(H(Hαα and Hand Hββ trade places)trade places)
“decouples”“decouples” hydrogens.hydrogens.
VariableVariable
δ
s !s !
VariableVariable δ (concentration and moisture-dependent)(concentration and moisture-dependent)
andand no couplingno coupling !!

Coupling is restored on cooling, because protonCoupling is restored on cooling, because proton
exchange is “frozen” (on the NMR time scale).exchange is “frozen” (on the NMR time scale).
CCHH33OOHH
OOHH peak often broad; disappears on additionpeak often broad; disappears on addition
ofof DD22O to the sample (O to the sample (HH//DD exchange).exchange).

1313
C NMR SpectroscopyC NMR Spectroscopy
Only 1%Only 1% 1313
C in nature:C in nature: NoNo 1313
C—C—1313
CC
splitting,splitting, since chances of finding twosince chances of finding two
1313
C adjacent are small (~0.01%).C adjacent are small (~0.01%).
(For the same reason: No(For the same reason: No 1313
C—H seen inC—H seen in 11
H NMR.H NMR.
99% of sample is99% of sample is 1212
C).C).
LargeLarge JJss 1313
CC HH JJ ~~ 100-150100-150 HzHz
1313
CC CC HH JJ ~~ 5-105-10 HzHz
ButBut:: 1313
C—H visible inC—H visible in 1313
C NMRC NMR..

Triplet ofTriplet of
quartetsquartets
Quartet ofQuartet of
tripletstriplets

Coupling to H can be removed byCoupling to H can be removed by
“broad band” irradiation of all Hs“broad band” irradiation of all Hs
Hence:Hence: all lines are singlets !all lines are singlets !
averages Haverages Hαα//ββ

SymmetrySymmetry
CC77HH1414 isomersisomers
Number of Cs:Number of Cs: 55 55
44 44 33 11 77

Chemical shift rangeChemical shift range :: 200 ppm200 ppm ((11
H: 10 ppm)H: 10 ppm)
Rules for deshielding same as inRules for deshielding same as in 11
H NMRH NMR
(multiply by a factor of 10-20)(multiply by a factor of 10-20)

Advanced TechniquesAdvanced Techniques
Distortionless polarization transferDistortionless polarization transfer
(“(“DEPTDEPT”)”) 1313
C NMR spectrum. Tells usC NMR spectrum. Tells us
whether the carbon is attached towhether the carbon is attached to 33,, 22,,
11 oror no hydrogenno hydrogen, i.e., i.e. CHCH33,, CHCH22,, CHCH, or, or CC..
Requires running the spectrum in threeRequires running the spectrum in three
different ways (using specific laser pulsedifferent ways (using specific laser pulse
sequences).sequences).
Example :Example : LimoneneLimonene..

Normal spectrumNormal spectrum
DEPT-90 spectrum:DEPT-90 spectrum: only CHonly CH
DEPT-135 spectrum:DEPT-135 spectrum: CHCH andand CHCH33 positivepositive
CHCH22 negative peaksnegative peaks

Two Dimensional NMRTwo Dimensional NMR
Correlated spectroscopy (Correlated spectroscopy (COSYCOSY).).
H/H or H/CH/H or H/C
11
H NMRH NMR
11
HH
NMRNMR
10 ppm10 ppm00
1010
CoupledCoupled
HsHs
11
HH
1313
CC
00 1010
200200
C—HC—H
connectivityconnectivity

MRI : Magnetic ResonanceMRI : Magnetic Resonance
ImagingImaging
Based on “relaxation” timesBased on “relaxation” times ββ αα, “, “TT11””
TT11 values differ with tissuevalues differ with tissue
(environment)(environment)
Whole body NMRWhole body NMR

MRI of Human AbdomenMRI of Human Abdomen
spleenspleenkidneyskidneys
liverliver

MRI, median sagittal section: cervicodorsal syringomyelia. This
condition is characterized by the presence of fluid-filled cavities
in the spinal cord substance.

Brain Tumor About to beBrain Tumor About to be
ZappedZapped

ThisThis spiralspiral
represents therepresents the 2323
stagesstages occurring inoccurring in
thethe firstfirst trimestertrimester
of pregnancy andof pregnancy and
every two weeksevery two weeks ofof
thethe second andsecond and
thirdthird trimesters.trimesters.

Stage 1Stage 1
FertilizationFertilization
1 Oocyte, 3001 Oocyte, 300
Million Sperm, 24 hoursMillion Sperm, 24 hours
0.1 - 0.15 mm0.1 - 0.15 mm
1 day post-1 day post-
ovulationovulation
FertilizationFertilization
begins when a spermbegins when a sperm
penetrates an oocytepenetrates an oocyte
(an egg) and it ends with(an egg) and it ends with
the creation of thethe creation of the
zygote. The fertilizationzygote. The fertilization
process takesprocess takes
about 24 hours.about 24 hours.
Stage 5Stage 5
ImplantationImplantation
Complete,Complete,
PlacentalPlacental
CirculationCirculation
System BeginsSystem Begins
0.1 – 0.2 mm0.1 – 0.2 mm
7-12 days post-7-12 days post-
ovulationovulation
Stage 2Stage 2
Stage 10Stage 10
CleavageCleavage
First Cell Division,First Cell Division,
Blastomeres,Blastomeres,
Mitotic divisionMitotic division
0.1-0.2 mm0.1-0.2 mm
1.5-3 days post-1.5-3 days post-
ovulationovulation
Neural Folds BeginNeural Folds Begin
to Fuse, Heartto Fuse, Heart
Tube FusesTube Fuses
1.5-3.0 mm1.5-3.0 mm
21-23 days post-21-23 days post-
ovulationovulation

Cross Sections of 3-DCross Sections of 3-D
ImageImage
…… of a humanof a human
embryo,embryo, 4444
days afterdays after
conceptionconception..
Roughly theRoughly the
size of a navy bean, it stillsize of a navy bean, it still
has webbed fingers andhas webbed fingers and
toes, but is alreadytoes, but is already
developingdeveloping a brain with twoa brain with two
hemisphereshemispheres,, the precursorsthe precursors
of vertebraeof vertebrae (dashlike(dashlike
structures in right slice) andstructures in right slice) and
internal organsinternal organs..

Stage 15Stage 15
Stage 23Stage 23
((6 to 8 weeks post fertilization6 to 8 weeks post fertilization))
Lens vesicle, nasal pit, hand plate;Lens vesicle, nasal pit, hand plate;
trunk widens; future cerebraltrunk widens; future cerebral
hemispheres distincthemispheres distinct
Head and neckHead and neck
((approximately 56-57approximately 56-57
postovular dayspostovular days))
Essential external andEssential external and
internal structures completeinternal structures complete

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