2D NMR spectroscopy

INTRODUCCIÓN A LA RESONANCIA MAGNETICA NUCLEAR BIDIMENSIONAL

• So far we have been dealing with multiple pulses but a single dimension - that is, 1D
spectra. We have seen, however, that a multiple pulse sequence can give different spectra
which depend on the delay times we use.

• The ‘basic’ 2D spectrum would involve repeating a multiple pulse 1D sequence with a
systematic variation of the delay time tD, and thentD1
plotting everything stacked. A very simple
example would be varying the time before acquisition:

tD2

… tD3

…
tDn

• We now have two time domains, one that appears during the acquisition as usual, and
one that originates from the variable delay. 1
2D NMR basics
• There is some renaming that we need to do to be more in synch with the literature:

• The first perturbation of the system (pulse) will now be called the preparation of the
spin system.
• The variable tD is renamed the evolution time, t1.
• We have a mixing event, in which information from one part of the spin system is
relayed to other parts.
• Finally, we have an acquisition period (t2) as with all 1D experiments.
cuando adquirimos ese
• Schematically, we can draw it evento artificial, que
like this:
creamos, origen de la fin, la adquisición es
segunda dimension conocida pero t2 no

Preparation Evolution Mixing Acquisition

Inicio del experimento aqui es donde

primer pulso que t1 se translada t2
perturba el estado la información
• t1 is the variable delay time, and NUEVO
de la muestra
t2 is the normal acquisition time. We can envision having
f1 and f2, for both frequencies…

• We’ll see that this format is basically the same for all 2D pulse sequences and
experiments. 2
 90y 90x

t2
t1
preparación
tiempo de
pulso 90
evolución
nos tira la
magnetización tiempo de adquisición

Starting before the second p / 2 pulse:

y z

90x x
x
o
y

y z

90x x
x
o
y

3
4
 90y 90x

t2
y z t1

90x x
x
o
y

y z

• The x axis component is not

90x x affected, but its amplitude will depend
x
o on the frequency of the line.
y

y z A(t1) = Ao x cos(o x t1 )

90x x
x
o
y

y z

o
90x x
x
y
5
stacked plot

A(t1)

t1
t1

o

f2 (t2)
Transformada de furier con el
tiempo de evolución

• Don’t forget T1 and T2 relaxation.

a medida que el tiempo de evolución cambia, tambien cambia las señales que se reflejan

6
A rudimentary 2D experiment (another view)

aqui se muestra como van cambiando las señales, en funcion del tiempo de evolución y el movimiento de los vectores7

t1 no se puede alargar hasta el infinito

esta honda poco a poco a ir disminuyendo, es como generar un fi artificial, POR EL TIEMPO DE RELAJACIÓN T1 Y T2 8
9
We have a cross-peak where the two lines intercept in the 2D map,in this case on the diagonal.
si se realiza la transfornada de furier con el tiempo de evolución t1

o

o
f1
espectro tridimensional

f2
la frecuencia va a ser la de mi señal

10
 contour plot.
o

o

o
f1 o
f2
f1

f2

el esquema en realidad es tridimensional pero nosotros lo pasamos a bidimensional para interpretarlo como un diagrama de contornos

11
12
 O

pulegone (COSY)

t1
time - time t2

time - frequency
t1
f2

frequency - frequency

f1
f2 13
contour-plot

f1

f2

off-diagonal peaks??

14
los experimentos INEPT / DEPT se basaba en la transferencia de magnetización (población)

Remember INEPT/DEPT: Transfer magn. From 1H to 13C through 1H-13C coupling

Heteronuclear Coherence Transfer

By a 90º --- tº ---90º---acquire sequence we’ll get Homonuclear Coherence Transfer

15
16
 Polarization transfer. SPT and SPI (OLD STUFF)
90
180s
selective polarization transfer, or SPT.

3,4

la inversion selectiva en una


de las señales afecta a la otra
que esté acoplada a ella
4 2,4
I S
 •2•
1,2
••••
3

S I
••
1


1,3
selective population inversion, or SPI
17
Homonuclear correlation - COSY
• COSY : COrrelation SpectroscopY, → homonuclear correlation spectroscopy.
• In our development of the 2D idea we considered an isolated spin.

 J (Hz)

I S
 • • ••

S I
I S
••••


Effect of the COSY 90y - t1- 90y - t1 pulse sequence

on a pair of coupled spins.
3,4
cuando realizamos lo nuevo, lo
del tiempo de evolucion, los

4
 2,4
vectores y las señales se
comportan muy similar a cuando
I S realizamos una inversion
selectiva SPI
•• 1,2
 ••••
2
3

S I
••
1

18
1,3
 90y 90x
A rudimentary 2D experiment (just a few minutes ago)
t2
y z t1

90x x
x
o
y

y z

90x x
A(t1) = Ao x cos(o x t1 )
x
o
y

y z

90x x
x
o
y

y z

o
90x x
x
y
19
• After the first p / 2, → two I vectors in the x axis, one moving at I + J / 2 and the other at
I - J / 2. The effect of the second pulse is that it will put the components of the
magnetization aligned with y on the -z axis, which means a partial inversion of the I
populations.

Considering that we are on-resonance with one of the lines and if t1 = 1 / 4 J:

z
y

90y x
x

y
J/2

20
21
• If we do it really general (nothing on-resonance), we would come to this relationship for
the change of the S signal (after the p / 2 pulse) as a function of the I resonance frequency
and JIS coupling:

AS(t1,t2) = Ao x sin( I x t1 ) x sin (JIS x t1 ) x sin( S x t2 ) x sin (JIS * t2 )

• After Fourier transformation on t1 and t2 , and considering also the I spin, we get:

S I

I

S
f1

f2

• This is the typical pattern for a doublet in a phase-sensitive COSY. The sines make the
signals dispersive in f1 and f2.
22
COSY (Jeener exp.)
magnitude mode

23
24
 Window functions (OLD STUFF)
se usa para los espectros del carbono 13, porque se necesita aumentar la relacion de señal/ruido

ganamos en señal/ruido pero

perdemos en resolución

F(t) = 1 * e - ( LB * t ) or F(t) = 1 * e - ( t / t )

se combinan estas dos señales para obtener la señal final (afilar el fi), se elimina parte del ruido inicial
para obtener mayor informacion inicial que es la referente al fi
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Other useful window functions
• Gaussian/Lorentzian: Improves resolution and does not screw up sensitivity as bad
as resolution enhancement alone.

F(t) = e - ( t * LB + 2 t2 / 2 )

• Hanning: Another resolution/sensitivity enhancement combo.

F(t) = 0.5 + 0.5 * cos( p t / tmax )

• Cosine - shifted cosine: Employed mostly for 2D spectra.

F(t) = cos( p t / tmax )

• The right window function depends on the experiment, and, as usual, there is a lot of
fooling around involved... 26
27
 Quadrature Detection

In order to distinguish the sign of frequencies, NMR signals must be collected along both the x
(e.g. cosine) and y (e.g. sine) axes. This is called quadrature detection. The signal is treated as a
“real” part (cosine) and an “imaginary” part (sine). They are both equally real and important,
but this allows easy mathematical manipulation from Euler’s relations:

Cos(t ) + iSin(t ) = eit Cos(t ) − iSin(t ) = e −it

real imaginary real imaginary

NMR Receiver

28
 PH = 0
Quadrature
detection S F
• How can we tell
which frequency S
is going faster or

PH = 90
slower relative to  (B1)
the carrier? The F
trick is to put 2
receiver coils at
90 degrees (with
a phase shift of
90 degrees) of PH = 0
each other:
F S

• The phase of the

faster signals is
opposite to that
of the slower PH = 90
signals, and the
computer is then F S
able to sort this
out.
29
 Spectrum phasing-Quadrature
detection
FT of cosine function FT of sine function
• The phase of the lines appears due to the
5
4 contribution of absorptive or real (cosines)
4

3 +
2
and dispersive or imaginary (sines)
2
20 40 60 80 100 120 components of the FID. Depending on the
-2
1
-4 relative frequencies of the lines, we’ll have
more or less sine/cosine component
Sum

10
S()x = S cosines() - real spectrum
=
S
8

6
S()y = sines() - imaginary
4

2
spectrum

• What we want is the purely absorptive

spectrum, so we combine different
amounts of the real (cosine) and the
imaginary (sine) signals obtained by the
detector. The combination depends on the
frequency of the spectrum:

S() = S()x + [ fo + f1() ] · S()y

• fo is called the zero order phase and f1

the first order phase. 30
Quadrature Detection in Two Dimensions

Quadrature detection in the detect dimension of two-dimensional experiments is

accomplished in the same way as in 1D experiments. The indirect detect dimension, t1, is similar.
Essentially there are 2 choices:
1) Absolute Value or Magnitude Spectrum- No quadrature detection in t1, all t1 FIDs are real.
2) Phase sensitive Spectrum: States (or States-Haberkorn or SHR)- or TPPI (or the Redfield method)-
As in the standard quadrature detection mode in the detect dimension, there is a complex FID created
by incrementing the phase of one pulse by 90º and recording twice as many discreet t1 points, so
there is phase difference of 90º creating a real and imaginary FID for a complex FID in t1. The
spectral width in t1 should be set the same as the spectral width in t2 for a 1H/1H experiment or in the
same manner; the spectral width is the real value.

31
COSY (Jeener exp.)
magnitude mode

32
33
phase-sensitive COSY. The sines make the signals dispersive in f1 and f2.

S I

I

S
f1

f2

34
f1

f2

35
 By the way...

Remember FOLDING and ALIASING?

36
Spectral Aliasing. (a) An aliased peak (blue peak) appears at a position in the
spectrum that is exactly one spectral width from its real position (dotted peak). (b)
Spectral Aliasing with sign inversion
37
Spectral Folding. (a) A folded peak (blue peak) appears at the position mirrored
about the spectrum boundary, as if you would “fold” the spectrum at the
spectrum boundary. (b) Spectral Folding with sign inversion

38
39
40
41
42
siempre se necesita un punto
de anclaje ( algo conocido)

primero se va al proton mas

desapantallado, en este caso
es CH (2). En el grafico se
puede ver que la señal de CH
(2) está relacionada con la
señal CH2 (3).

A su vez, la señal del CH2 (4)

tambien está relacionada con
el CH2 (3)

43
 2H 1H HO OH
1H 1H 1H 3
4
1 2
N3
(CH3)2CHO2C O 5
6

iPr moiety is out of range

se empieza por el H2 porque es el que está

mas desapantallado y solo integra para uno,
solo tiene relacion con el H3. por lo que el 2
estaría a 4,6 pp, y el 3 a 4,3 ppm

Ahora el proton H3 está relacionado con el H4

por lo que el H4 estaría a 4 ppm pero hay que
tener en cuenta que en esa señal se
encuentran dos protones. H5 está acoplado
con H4 (por alguna razon que no entendí)
entonces la señal de H4 y H5 estan a 4,0 ppm

el H6 está a 3,6 ppm y H6' a 3,4 ppm, el proton

H5 está relacionado con ellos.

44
When the chemical shift differences Dd
between the coupled nuclei are small the
cross peaks, which are close to the
diagonal, are difficult to recognize, as this
is the region where the strong diagonal
peaks with their broad wings interfere.

If instead of the second 90ºx' a smaller

angle Qx' the transfer of magnetization
takes place preferentially and the peaks
width is reduced, thus avoiding mixing.

45
entre mayor numero de medidads de t1, mas tiempo va a durar el experimento y la resolución va a ser mejor 46
47
48
49
glutation

50
Long range COSY (also called Delayed-COSY). A fixed delay
D, usually about 0.1 to 0.4 s, is introduced before and after the
second 90ºx' pulse, so that the sequence becomes:

90ºx'--t1--D--90ºx'--D—FID

The effect of the delay is to allow the development of

correlation effects also for very weakly coupled protons, so that
in the 2D spectrum we find correlation peaks for protons that
show no detectable scalar couplings in the 1D 1H-NMR
spectrum.
Nowadays overcome by TOCSY

51
este no es cosy normal
sino cosy delay donde
se ve todo el sistema de
espin

52
53