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•In the 13C spectra of 13C-1Hn groups the signal will be split by large 1JCH.
•This splitting will reduce the sensitivity of signals which often are already naturally
weak
•Such a large splitting can also cause severe overlap of lines and result in complex
spectra.
e x c h a n g e / s p in s
- I C I H C
-I I
H
C y y y y
-Iy - I yC
C H
I I I C I H
x x x x
t = 0 t = t = t = 2
• Such an exchange of α/β states can be achieved by application of a 180̊ pulse on the
protons (remember that an 180̊ pulse inverts all spins). In the product operator
formalism, that is equivalent to reversing the sign of the I xCIzH term:
π
-IyC cos(JCHπτ) + IxCIzH sin(πJCHx τ) ─────> -IyC cos(JCHπτ) - IxCIzH sin(πJCHτ)
Practical application of decoupling
• The ideal solution would therefore be to apply a 180̊ proton pulse between each
sampled data point during acquisition.
• In order to achieve 180̊ pulse between every sampled data point many (32000)
strong pulses (τπ ≪ DW) would have to be applied in rapid succession.
• In a typical 13C spectrum (220 ppm at 75 MHz) DW ≈30 μs or less. That is
usually not achievable experimentally
/2 (x ) (y ) / x /2 (x ) (y ) / x
C P D d e c o u p lin g 1
w ith M L E V H
id e a l 1
d e c o u p lin g H
13
C
0 2
t ( a c q u is it io n )
Problems with achieving “ideal“
decoupling requirements
• duty cycle of amplifier
• damage to probe
• As DW ≪ 1/J (JCH ≈ 150 Hz, 1/J = 6.7ms) dephasing from J coupling is small during
DW, so inversion does not have to occur between every sampled point.
• Typically, decoupler pulses with τ90 = 100 μs are used (γB1/2π = 2.5 kHz).
Problem of Bandwidth of
decoupling
• Most basic decoupling scheme: continuous wave irradiation with a power of
γB1/2π = 2.5 kHz (cw decoupling)
• However, for more many proton signals over a shift range of 10 ppm to be
decoupled, another requirement is that γB1/2π needs to be strong enough to cover
that range
• CW decoupling can often not performed with the power required to achieve the
required bandwidth
Composite Pulse Decoupling
• It is more efficient to use a series of pulses with variable phases (composite
pulses): => Composite Pulse Decoupling (CPD)
• The effect is non trivial in origin, and will not treated fully from scratch here
Basic theory of NOE
• The observation of NOE enhancement is a special relaxation effect caused by the
change in population of the proton levels during the decoupling.
• For now we accept that in the presence of through space (dipolar) spin-spin
couplings, molecular motion induces random transitions between levels related by
flipping one or both coupled spins.
Transitions in a two spin system
W 2 W 0 c r o s s r e la x a tio n
W 1C W 1 H W 0 W 2 s p in la ttic e r e la x a tio n
1
T1
Nothing better than a picture
0
13
C 13
C
+
H C
+
H C
C
W
2
1 1
H H
W 0
1 1
H H
H+ C
+
H C
13 13
H + C
C C
1
13 1
13 1
13
H C H C H C
in itia l th e rm a l tu rn o n p ro to n e s ta b lis h n e w e q u ilib riu m
e q u ilib riu m d e c o u p lin g u n d e r d e c o u p lin g c o n d itio n s
P o p u la tio n d iffe re n c e s :
1
H : 0
} H 0
13
C :
} > > W 1: /2 +
1 H C
C C if W 2
= C[ 1 + H /( 2 C)]
m s = 2 : H + C C (n o t lo n g e r
m s = 0 : H - C C a t e q u ilib riu m )
Thus the carbon signal will increase up to a factor of three in intensity upon
decoupling of protons:
M0C ∼ ΔC MCNOE ∼ (ΔC + ΔH/2) or MCNOE = MC0 + η M0C
• W0 and W2 depend only on dipolar interaction between 1H and 13C, whereas W1H and
W1C may also include other contributions to the relaxation (Chemical shift anisotropy,
paramagnetic impurities like oxygen), so the theoretical enhancement will not always
be observed.
• Note that dipolar coupling does not require a bond, but directly bound protons are also
close in space.
Practical considerations of NOE for
carbon NMR
• Distance dependence: Tertiary carbons like C=O and >C< show only very little
enhancement and show up weaker than protonated carbons => intensities will not be
proportional to number of C-atoms
• additional mechanisms of relaxation will only contribute to W1 and may reduce NOE
(Chemical shift anisotropy, unpaired electrons from oxygen)
• Nuclei with negative will exhibit a negative NOE, which in some cases may decrease
signal (15N: N-15 -1/10 H, 113Cd)
• large molecules (M > 3000): W2CH ≈ W0CH and ήmax ≈ 0.25 (small NOE)
• NOE will depend on molecular size and will be close to zero for macromolecules.
• As NOE is a relaxation phenomenon it will take time to build up after turning the decoupler
on.
Basic multiple pulse experiments
Quantitative 13C NMR
•Goal: get decoupled spectrum without (or with minimum) NOE:
•Solution: NOE takes time to build up (up to a few seconds). On the other hand
decoupling occurs instantaneously after turning on decoupling:
•decouple only during aq, not during d1: “inverse gated decoupling” (zgig)
• Solution: Decouple during d1, but not during aq:“gated decoupling” (zggd)
• This will not solve the problem of spreading signal intensity over many multiplet
lines and potential overlap in crowded spectra, but it gives improved sensitivity
over simple one pulse experiment.
Overview of different decoupling
schemes
d e c o u p le
Regular decoupling
d1
p1
Gated Decoupling
• Consider an experiment where we add a time τ = 1/1JCH between the 13C excitation
pulse and the start of acquisition with decoupling:
1
H C PD
o
90 x
13 1
C = /J
Evolution of spins under coupling
after a pulse:
C
C H
C H 2
C H 3
1
t 0 1
/4J /2J 1
/J
Regard a C-Hx fragment: Evolution of 13C x,y -magnetization with 1H
coupling after time t
After τ = 1/J all spins will again be aligned, but their relative phases will
depend on multiplicity.
Obtaining multiplicity edited spectra
in real samples
• On first sight such an experiment looks like a good multiplicity edited spectrum.
• Why will this not work in a real experiment ?
• Above experiment does not take into consideration rotation of spins due to chemical
shift, so it would only work for one spin exactly on resonance.
• For many different spins, dephasing will occur during τ also for different Ω values
as Ω >> πAJ.
• removal of chemical shift evolution necessary during τ:
1
H
o
90 x
13 1 1
C d1 = /J = /J
c re a te N O E d e fo c u s s p in s re fo c u s c h e m ic a l a c q u ir e F ID
a c c o rd in g to s h if t d iff e re n c e s ,
m u ltip lic ity s to p c o u p lin g
1 1
/J /J
APT without refocusing APT with refocusing of
of chemical shift chemical shift
Notes on the APT experiment
• It is important to turn the decoupler on during the second τ period. With
decoupling on the coupling will be “frozen” after the first τ and only chemical shift
will refocus. With decoupling off during second τ also coupling will be refocused
(it will also act as a decoupling pulse).
• Note that the condition τ = 1/1JCH can not perfectly achieved for all carbons as the
coupling constants can vary (typically 125 - 160 Hz) and non-perfect editing will
occur.
Note: Spectra can be phased arbitrarily, so it can be either CH, CH 3 “up”, C, CH2
“down” or CH,CH3 “down”, C, CH2 “up”. The command apk can give you thus
either result as the computer itself does not know or care whether a signal is CH or
CH2.
Typical use of 180̊ pulses
o
180x
magnetization. 13
A N D c h e m ic a l s h ift
o
180x
1
H
re m o v e c h e m ic a l s h ift
o o k e e p C H c o u p lin g
90x 180x
13
C
• Can we do better ?
Consider 13C-1H fragment: H
H (C -) H
H
a b c d
z z z z
I zC I H
- I xC I H
y
x y x y x y
x C H
I x C I H Iz I
H
-M 0 I yH -M 0
H
I xH I zC -M 0
H
I zH I zC -M 0
H
I yC I zH
C
-M 0 I zC -M 0
C
I zC -M 0
C
I yC -M 0
C
I yC
Accounting for chemical shift
dispersion
• This sequence will work for one C-H if pulses are applied on resonance (ΩC and ΩH
= 0).
• In real life however many peaks will be present and additional rotation by Ω H will
occur during τ = 1/2 J . Thus the vectors will not be aligned along x at point b.
• However, insertion of two 180̊ pulses in the center of τ will remove the chemical
shift dephasing but keep the coupling evolution:
CH CH2 CH3
|
|
|
| | | |
| J | J J | J | J J
))))))) ) ) ) ) ) ) ) |) ) ) ) ) ) ) ) ) ) ) )) ) ) )) ) ) ) ) ) ) )
| | | |
| | | |
|
• One disadvantage of the basic INEPT is that the multiplets appear in anti phase
( /2 )x ( /2 )y
1
H
2 2 C PD
( /2 )y
13
C t2
2 2
• Note that during τ one considers a proton coupled to one carbon, while during Δ
one has to consider a carbon coupled to one, two or three protons
Editing using INEPT
• The situation in INEPT during the second refocusing is similar, but not identical to
APT (one starts with anti phase magnetization at the beginning of Δ)
• note that non-protonated carbons will not show up in that experiment as all
magnetization arizes from initial proton magnetization
Editing with INEPT
x
y re fo c u se d IN E P T :
1 --- ---
H a 2 b c CPD e d itin g w ith d e la y
2
J : C H o n l y
= 3 /( 4 J ): C H ,C H 3 u p , C H 2 d o w n
= 1 /(4 J ): C H , C h 2 , C h 3 p o s itiv e
13
C d e
d iffe re n c e s in J v a lu e s e ffe c t p h a s e s
d e c o u p lin g d u r in g o f s ig n a ls
a c q u is itio n
H H H C H
a )-M 0 Iy b )-M 0
H
I xH I zC c )-M 0
H
I zH I zC d )-M 0 Iy Iz e )M 0
H
I xCs in ( J C H ) (C H )
s in ( J C H ) c o s ( J C H ) ( C H 2)
s in ( J C H ) c o s ( J C H ) ( C H 3)
2
Editing using DEPT
• Disadvantage of editing with refocussed INEPT: As in APT it works perfect only if
all coupling constants 1JCH are the same. Typical values vary from 125 Hz - 150 Hz
(up to 200 Hz in some cases)
• The problem is the effect of the first 13C π/2 pulse which converts IxHIzC into -IxHIyC.
This is called a double/zero quantum coherence.
• The quality of editing in the DEPT does not depend on the accuracy of matching
the delay τ to the coupling constant but is determined by the pulse width β and
depends on the different operators generated by different numbers of protons
coupled to the 13C.
Editing with INEPT/DEPT:
Dependence on delay/pulse