Download NMR Spectroscopy

NMR Spectroscopy
Applications
Drug design
MRI
Food quality
Metabonomics
Structural biology
NMR Spectroscopy
Basic Principles
N.M.R. = Nuclear Magnetic Resonance
Spectroscopic technique, thus relies on the interaction between material and electromagnetic radiation
The nuclei of all atoms possess a nuclear quantum number, I. (I0, always multiples of .)
Only nuclei with spin number (I) >0 can absorb/emit electromagnetic radiation.
Even atomic mass & number: I = 0 (12C, 16O)
Even atomic mass & odd number: I = whole integer (14N, 2H, 10B)
Odd atomic mass: I = half integer (1H, 13C, 15N, 31P)
The spinning nuclei possess angular momentum, P, and charge, and so an associated magnetic moment, .
= x P
Where is the gyromagnetic ratio
NMR Spectroscopy
Basic Principles
B0
The spin states of the nucleus are quantified:
I, (I - 1), (I - 2), … , -I
I= (e.g. 1H)
Energy
–
–
E=h=hB0/2
B0=0
B0>0
NMR Spectroscopy
Basic Principles
In the ground state all nuclear spins are disordered, and
there is no energy difference between them. They are
degenerate.
Bo
Since they have a magnetic moment, when we apply a
strong external magnetic field (Bo), they orient either
against or with it:
There is always a small excess of nuclei (population
excess) aligned with the field than pointing against it.
NMR Spectroscopy
Basic Principles
Bo>0
Bo=0
E=h0=hB0/2
0 is the Larmor Frequency
0=B0, angular velocity
z
B0
y
x
NMR Spectroscopy
Basic Principles
Each level has a different population (N), and the difference between the two is related
to the energy difference by the Boltzmman distribution:
N/N = eE/kT
E for 1H at 400 MHz (B0 = 9.5 T) is 3.8 x 10-5 Kcal/mol
N/N =1.000064
The surplus population is small (especially when compared to UV or IR).
That renders NMR a rather insensitive technique!
NMR Spectroscopy
The electromagnetic spectrum
-rays
600
1022
1020
Mossbauer
500
400
X-rays
visible 1014
infrared
electronic
100
rotational
108
NMR
106
/Hz
4
200
aldehydic
aromatic
6
31P
vibrational
1010
radiofrequency
8
2
0
1012
microwave
19F
300
1018
ultraviolet 1016
10
1H
13C
/MHz
olefinic
acetylenic
aliphatic
/ppm
NMR Spectroscopy
The Vector Model
M0
y
x
y
x
y
y
x
x
NMR Spectroscopy
NMR excitation
z
B1 = C * cos (ot)
Mo
y
B1
x
Bo
i
Transmitter coil (x)
B1 is an oscillating magnetic field
y
y
-0
+0
x
x
NMR Spectroscopy
Laboratory vs. Rotating frame
z
z’
+0
M0
M0
y
+0
x
y’
B1
-0
Laboratory frame
x’
-20
Rotating frame
NMR Spectroscopy
Effect on an rf pulse
z
z
M0
=1tp (degrees)
=90°
B1
x
y
y
B1
x
=180°
NMR Spectroscopy
Magnetization properties
1H=400,000,000 Hz
A=400,000,005 Hz
z
A
y
y
B1
MAy
MAx
=5 Hz
x
x
y
x
x
y
x
y
x
y
NMR Spectroscopy
Magnetization properties
1H=400,000,000 Hz
A=400,000,005 Hz
z
A
B1
MAy
y
y
y
MAx
=5 Hz
x
x
x
MAy=MA cost
detector
MAx=MA sint
IM
IM
MA
MA
t
t
t
NMR Spectroscopy
The Fourier Transform
FT
time domain
IM
frequency domain
=1/t
t
FT
t
time
frequency
NMR Spectroscopy
The Fourier Transform
IM
Signal Induction Decay (FID)
time
NMR Spectroscopy
The Fourier Transform
FT
NMR Spectroscopy
Continuous wave vs. pulsed NMR
o or Bo
o or Bo
time
NMR Spectroscopy
Continuous wave vs. pulsed NMR
• For cos(t)
FT
absorptive lines
• For sin(t)
FT
despersive lines
NMR Spectroscopy
Continuous wave vs. pulsed NMR
A monochromatic radiofrequency pulse is a combination of a wave
(cosine) of frequency 0 and a step function
*
=
tp
Since f=1/t, a pulse of 10 s
duration excites a frequency
bandwidth of 105 Hz!
FT
o
NMR Spectroscopy
Continuous wave vs. pulsed NMR
E t ~ h or t ~1
NMR Spectroscopy
Single-channel signal detection
z
y
x
FT
+
+
0
-
NMR Spectroscopy
Quadrature detection
z
y
x
My
Mx
My
Mx
+
NMR Spectroscopy
Quadrature detection
cos
y
sin
x
+
0
+
+
-
0
Hz
Hz
NMR Spectroscopy
The Chemical Shift
The NMR frequency of a nucleus in a molecule is mainly determined by its
gyromagnetic ratio and the strength of the magnetic field B
The exact value of depends, however, on the position of the nucleus in the molecule or
more precisely on the local electron distribution
this effect is called the chemical shift
NMR Spectroscopy
The Chemical Shift
E=h=hB/2
Nuclei, however, in molecules are never isolated from other particles that are charged
and are in motion (electrons!).
Thus, the field actually felt by a nucleus is slightly different from that of the applied
external magnetic field!!
NMR Spectroscopy
The Chemical Shift
E=h=hBe/2
Beff, is given by B0-B= B0-B0=B0(1-)
=
and is the chemical shift
=
(-ref)
ref
B0(1-)
2
106 106 (ref-)
NMR Spectroscopy
The Chemical Shift
methyl
protons
amide protons
protons
methylene
protons
aromatic ring
750 MHz 1H spectrum of a small protein
shielding
frequency
magnetic field
NMR Spectroscopy
The Chemical Shift
NMR Spectroscopy
The Chemical Shift
NMR Spectroscopy
Nuclear Shielding
=dia + para + nb + rc + ef + solv
diamagnetic contribution
paramagnetic contribution
neighbor anisotropy effect
ring-current effect
electric field effect
solvent effect
NMR Spectroscopy
Nuclear Shielding - diamagnetic contribution
The external field B0 causes the electrons to circulate within their orbitals
B0
hB0
hB0(1-)
B’
The higher is the electron density close to the nucleus, the larger the protection is!
NMR Spectroscopy
Nuclear Shielding - diamagnetic contribution
Depends on the electronegativity
CH3X
NMR Spectroscopy
Nuclear Shielding - paramagnetic contribution
The external field B0 mixes the wavefunction of the ground state with that of the excited state
The induced current generates a magnetic field that enhances the external field and deshields the
nucleus
LUMO
B0
HOMO
p =
1
1
R3
NMR Spectroscopy
Chemical shift range
1H;
~10 ppm
13C;
~200 ppm
19F;
~300 ppm
31P;
~500 ppm
Local diamagnetic and paramagnetic currents make only modest contributions to 1H shielding!
NMR Spectroscopy
Chemical Shift Anisotropy
Nuclear shielding, , is a tensor.
The distribution of the electrons about the nucleus is non-sperical- thus, the magnitude of the
shielding depends on the relative orientation of the nucleus with respect to the static field.
In isotropic cases: = (11 + 22 + 33)
In static cases, e.g. solid state
NMR Spectroscopy
Nuclear Shielding - neighboring group
B0
B
A
B
μpar > μper
A
μpar < μper
+
+
+
-
-
+
NMR Spectroscopy
Nuclear Shielding - neighboring group
+
-
C
C
-
μpar > μper
C
+
μpar < μper
+
+
C
-
C 2H 4
7
6
C 2H 2
5
4
3
C 2H 6
2
1
0
ppm
NMR Spectroscopy
Nuclear Shielding - ring-current effect
More pronounced in aromatic rings due to the electron clouds
Bo
9.28
e-
-2.99
NMR Spectroscopy
Nuclear Shielding - hydrogen bonding
Hydrogen bonding causes deshielding due to electron density decrease at the proton site
[EtOH] in CCl4
CH3
CH2
OH
1M
0.1M
0.01M
0.001M
6
4
2
0
ppm
NMR Spectroscopy
Spin-spin (scalar) coupling
HF (1H-19F)
H
F
JHF
JHF
NMR Spectroscopy
Spin-spin (scalar) coupling
HF (1H-19F)
H F
H
H F
Bo
19F
1H
H F
H
H F
19F
1H
Nuclear moment
Magnetic polarization
of the electron
E=h JAX mA mX
where m is the magnetic quantum number
JAX is the spin-spin coupling constant
NMR Spectroscopy
Spin-spin (scalar) coupling
AX2
AMX
AX3
NMR Spectroscopy
Spin-spin (scalar) coupling
Strong coupling – <10|J|
NMR Spectroscopy
Spin-spin (scalar) coupling
The principal source of scalar coupling is an indirect interaction mediated by electrons involved in chemical bonding
The magnitude of interaction is proportional to the probability of finding the electron at the nucleus (R=0)
Magnitude in Hz- independent of the external magnetic field
H3C – CH3
H2C – CH2
HC CH
125 Hz
160 Hz
250 Hz
NMR Spectroscopy
Spin-spin (scalar) coupling
Three-bond coupling most useful since it carries information on dihedral angles
Empirical relationship: the Karplus relation
3J
= A + B cos + C cos2 NMR Spectroscopy
Chemical shifts on the rotating frame
500 MHz
z
3
y
2
0
x
t
z
y
x
=500 Hz
NMR Spectroscopy
Spin couplings on the rotating frame
J
z
y
0
x
t
z
=-J/2 Hz
y
x
=+J/2 Hz
NMR Spectroscopy
The basic spin-echo pulse sequence
90° applied
along x axis
180° applied
along y axis
x
delay
y
acquisition
NMR Spectroscopy
90° applied
along x axis
Effect of spin echo on chemical shift evolution
180° applied
along y axis
x
y
acquisition
delay
z
z
y
90x
y
x
x
z
z
y
z
y
x
180y
A
x
y
x
A
NMR Spectroscopy
90° applied
along x axis
Effect of spin echo on scalar coupling evolution
180° applied
along y axis
x
y
acquisition
delay
z
z
y
90x
1H-X
y
x
x
z
z
-J/2
y
z
(only
180y
y
y
-J/2
x
+J/2
x
+J/2
x
1H)
NMR Spectroscopy
90° applied
along x axis
Effect of spin echo on scalar coupling evolution
180° applied
along y axis
x
y
acquisition
delay
z
z
y
90x
1H-X
y
x
x
z
z
-J/2
z
180y
y
y
+J/2
x
+J/2
x
-J/2
x
y
(both 1H and X)
NMR Spectroscopy
Water suppression by the Jump and Return method
z
z
y
90x
y
x
x
z
z
y
z
y
A
90-x
x
y
x
x
NMR Spectroscopy
Water suppression
NMR Spectroscopy
Spin decoupling
decouple
1H
13C
H C
H
H C
H C
H
H C
NMR Spectroscopy
The J-modulated spin echo
decouple
1H
y
x
13C
NMR Spectroscopy
The J-modulated spin echo
decouple
1H
y
x
13C
NMR Spectroscopy
The J-modulated spin echo
If =180J degrees
C:
CH:
CH2:
CH3:
I=1
I cos
I cos2
I cos3
decouple
1H
y
x
13C
NMR Spectroscopy
The J-modulated spin echo
decouple
1H
y
x
13C
=1/J
13C
(ppm)
NMR Spectroscopy
Sensitivity enhancement
NMR has poor sensitivity compared to other analytical techniques
The intrinsic sensitivity depends upon the gyromagnetic ratio, A greater contributes to:
a high resonant frequency- large transition energy difference- greater Boltzmann population difference
high magnetic moment and hence a stronger signal
high rate of precession which induces a greater signal in the detection coil
So, the strength of NMR signal is proportional to 3
Noise increases a square-root of observed frequency
}
S/N
5/2
NMR Spectroscopy
Sensitivity enhancement by polarization transfer
Signal sensitivity enhancement by transferring the greater population differences of high spins onto their spin-coupled low- partners.
C2
1H-13C
H2
H1
C1
spin pair
NMR Spectroscopy
Sensitivity enhancement by polarization transfer
Signal sensitivity enhancement by transferring the greater population differences of high spins onto their spin-coupled low- partners.
C
2C
C2
H2
+C
2
2
H1
C2
H2
+C
2
C
C1
C
2+2C
2
H1
C1
(inverted)
2C
C
2+2C
+C
+C
C1
H1
H1
H2
C1
C2
H2
C2
-3:5
NMR Spectroscopy
Sensitivity enhancement by polarization transfer
Signal sensitivity enhancement by transferring the greater population differences of high spins onto their spin-coupled low- partners.
coupled
decoupled
INEPT
refocused
INEPT
refocused,
decoupled
INEPT
NMR Spectroscopy
Relaxation
When perturbed, the nuclear spins need to relax to return to their equilibrium distribution
E.g. when the sample is put into a magnet, the Boltzmann distribution of spins among the energy
levels changes due to a change in the energy of the various levels
E.g. after applying electromagnetic radiation, which induces transitions between energy levels,
the system returns to its equilibrium
This process is called relaxation
NMR Spectroscopy
Longitudinal Relaxation: Establishing Equilibrium
z
y
x
z
z
y
y
x
x
z
z
x
x
NMR Spectroscopy
Longitudinal Relaxation: Establishing Equilibrium
Recovery of the z-magnetization follows exponential behavior
dMz
dt
=
(M0-Mz)
T1
Mz=M0 (1-2e-t/T1)
where T1 is the longitudinal relaxation time
NMR Spectroscopy
Longitudinal Relaxation: Measurement
x
x
z
z
z
180x
z
y
x
90x
y
x
y
y
x
x
z
z
90x
y
x
y
x
NMR Spectroscopy
Longitudinal Relaxation: Measurement
x
x
NMR Spectroscopy
Longitudinal Relaxation: Exponential growth
Mz=M0 (1-2e-t/T1)
By the end of 5T1 sec, the magnetization has recovered by 99.33%
NMR Spectroscopy
Longitudinal Relaxation: optimizing sensitivity
NMR Spectroscopy
Longitudinal Relaxation: optimizing sensitivity
NMR Spectroscopy
Longitudinal Relaxation: optimizing sensitivity
optimum pulse repetition time when using 90º
Quantitative measurements and integration
NMR Spectroscopy
Transverse Relaxation: magnetization loss in the x-y plane
y
x
-
-
-
y
y
y
+
+
+
x
x
time
x
NMR Spectroscopy
Transverse Relaxation: magnetization loss in the x-y plane
=
1
T2*
NMR Spectroscopy
Transverse Relaxation: Measurement
x
z
z
z
90x
-
y
y
y
x
+
x
z
180y
+
x
y
z
y
y
x
x
NMR Spectroscopy
Transverse Relaxation: Measurement
NMR Spectroscopy
T1 vs T2 Relaxation
T1 T2
For small molecules, T1 T2
For large molecules, T1 >> T2
Longitudinal relaxation causes loss of energy from the spins (enthalpic)
Transverse relaxation occurs by mutual swapping of energy between spins (entropic)
NMR Spectroscopy
Relaxation mechanisms
Nuclear spin relaxation is not a spontaneous process; it requires stimulation by
suitable fluctuating fields to induce the necessary spin transitions
Two main mechanisms
Dipole-dipole
Chemical shift anisotropy
NMR Spectroscopy
Relaxation mechanisms
Longitudinal relaxation requires a time-dependent magnetic field fluctuating at the Larmor frequency
The time-dependence originates in the motions of the molecule (vibration, rotation, diffusion etc)
Molecules in solution “tumble”. This “tumbling” can be characterized by a rotational correlation time c
c is the time needed for the rms
deflection of the molecules to be ~ 1
radian (60°)
NMR Spectroscopy
Spectral density function
Rotational diffusion in solution occurs at a range of frequencies
1/c ~ rms rotational frequency (radians s-1)
The probability function of finding motions at a given angular frequency can be described by the
spectral density function J()
NMR Spectroscopy
Spectral density function
Frequency distribution of the fluctuating magnetic fields
NMR Spectroscopy
Spectral density function: Longitudinal relaxation
Spins are relaxed by local fields fluctuating at the Larmor frequency 0
So, the relaxation rate (R1) will be proportional to the J(0)
1/T1= R1 = 2 <B2> J(0)
Knowing the form of J() we can predict the dependence of the spin-lattice relaxation time (T1=1/
R1) on the correlation time c for a given NMR frequency 0
0c=1 ,J(0) = c= 1/0
and T1 is minimum (R1 maximum)
0c<<1 (small molecules), J(0) ~ 2c and T1
decreases (R1 increases) with increasing c
0c<<1
0c>>1
(e.g.by decreasing the temperature)
0c=1
0c>>1 (large molecules), J(0) ~ 2/02c
and T1 increases (R1 decreases) with
increasing c (e.g. by decreasing the
temperature)
NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
Nuclei with non-zero quantum numbers have magnetic dipoles
They behave like small magnets and induce small magnetic fields that affect neighboring nuclei
Magnetic field, B, generated by a magnetic dipole NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
Representation of the dipolar magnetic field B, generated by a magnetic dipole lines of force
density plots
Bμz
Bμz is zero for =±54.7˚ (magic angle)
Bμx
NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
The z component of their dipole magnetic field will affect the field experienced by the
other nucleus and cause splitting
± sign refers to the quantum number of A (±)
X
A
Bμ
Thus, the splitting in the spectrum of X is
A
KAX vary with the distance
e.g. KCH is 9000 Hz at 1.5 Å and 30 Hz at 10 Å
NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
Splitting of the AX spectrum depends on In a crystal with fixed distances and angles the dipolar splitting vary with the crystal
orientation with respect to the external magnetic field
NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
Molecules in liquids rotate, “tumble” rapidly with typical frequencies between 1012 to
108 Hz for small molecules and proteins, respectively.
Those frequencies are much larger than typical dipolar couplings (105 Hz)
The angular part of the dipolar splitting is averaged over all possible orientation to 0
Although they are not directly observed in solution, dipolar couplings play an important
role in spin relaxation
The local field experienced at one nucleus as a result of its neighbor will fluctuate as the
molecule tumbles
NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
R1 depend of the gyromagnetic ratio of the nuclei (e.g. H-H relaxation more efficient than C-H)
NMR Spectroscopy
Relaxation mechanisms: Chemical shift anisotropy
The distribution of the electrons about the nucleus is non-sperical- thus, the magnitude of the
shielding depends on the relative orientation of the nucleus with respect to the static field.
As the molecule tumbles, it creates a fluctuating magnetic
field
NMR Spectroscopy
Nuclear Overhauser Effect (NOE)
NOE: change in intensity of one resonance when the spin transitions of another are perturbed
from their equilibrium populations
perturbation: saturation or inversion
The two spins should “communicate” through dipole-dipole interaction
NOE is observed for spin I when spin S is perturbed
NMR Spectroscopy
Nuclear Overhauser Effect (NOE)
Origin of the NOE
N-/2
N-
S
N
I
I
S
N
0
N/2
I
I
0
S
N+/2
N+
S
S
I
S
I
N+/2
NMR Spectroscopy
Nuclear Overhauser Effect (NOE)
Six possible transitions in a two-spin system
W1S
W1I
W2
W0
W1I
W1S
Only single transitions can by observed by NMR (W1)
W0 and W2 are cross-relaxation pathways, responsible for the NOE
NMR Spectroscopy
Nuclear Overhauser Effect (NOE)
N-/2
N-
S
N
I
N
0
N/2
I
S
S
I
I
S
S
I
S
I
S
N+/2
S
>
>
W2
I
0
W0
<
0
I
S
I
<
0
S
negative NOE
positive NOE
S
N+/2
0
N+
0
I
I
S
I
NMR Spectroscopy
Nuclear Overhauser Effect (NOE)
W1 tends to reduce the magnitude of the NOE
Saturating for a period of time that is long relative to the relaxation times allows a new
steady-state of populations to arise
IS, cross-relaxation rate: dictates the sign of the NOE
IS, dipolar longitudinal relaxation rate of spin I: it serves to reduce the magnitude
Thus, NOE is related to molecular motion!
NMR Spectroscopy
Nuclear Overhauser Effect (NOE)
1H
at 400 MHz
W1 at 400 MHz
W2 at 800 MHz (WI+WS)- stimulated by rapidly tumbling molecules
W0 at Hz-kHZ (|WI-WS|)- stimulated by slowly tumbling molecules
Small molecules exhibit positive NOEs
Large molecules exhibit negative NOEs
NMR Spectroscopy
Nuclear Overhauser Effect (NOE)
c=1.12
Variation in NOE as a function of molecular tumbling rates
NMR Spectroscopy
Field gradient
Bg
Variation of magnetic field strength along the z axis
NMR Spectroscopy
Field gradient
-!Bg
+!Bg
90º
-Bg, !g
Bg, !g
+!B
-!Bg
g
defocused
(dephased)
refocused
(rephased)
NMR Spectroscopy
Field gradient
x
RF
Gz
stronger gradient
NMR Spectroscopy
Field gradient
x
RF
Gz
Variation of the second gradient pulse (90 to 110% of the first)
NMR Spectroscopy
Diffusion-ordered spectroscopy
Bg
!!
x
!!
x
"!
#!
!!
"!
#!
G gradient strength
D diffusion coefficient
NMR Spectroscopy
Diffusion-ordered spectroscopy
mobility
NMR Spectroscopy
Multi-dimensional NMR
One dimension
Two dimensions
NMR Spectroscopy
Multi-dimensional NMR
To generate a spectrum with two frequency domains, f1 and f2, it is necessary to sample
data as a function of two separate time variables, t1 and t2.
General scheme for 2D NMR experiment
D
P: Preparation
E: Evolution
M: Mixing
D: Detection
P
E
t1
M
t2
NMR Spectroscopy
Multi-dimensional NMR
A
hv
B
NMR Spectroscopy
Multi-dimensional NMR
NMR Spectroscopy
COSY (COrrelated SpectroscopY)
Correlation through bonds (J-coupling)
NMR Spectroscopy
TOCSY (Total COrrelated SpectroscopY)
Correlation through bonds (J-coupling)
x
!m
t1
t2
spin-lock
JAB
A
!m
A
A
A
B
B
B
B
JBC
C
D
E
C
D
E
D
E
C
C
JCD
D
JDE
E
NMR Spectroscopy
COSY vs. TOCSY
Correlation through bonds (J-coupling)
COSY
TOCSY
NMR Spectroscopy
COSY vs. TOCSY
Correlation through bonds (J-coupling)
NMR Spectroscopy
General schemes for 2D NMR
Relative sensitivity
P
a)
H
E
H
t1
1H-13C
1H-15N
1
1
1
2.5
4
10
(traditional)
t2
H
t1
X
d)
1H-31P
t2
X
c)
D
t1
X
b)
M
4
8
30
10
32
300
t2
H
X
t1
t2
(inverse)
modern
NMR Spectroscopy
Heteronuclear Single Quantum Coherence (HSQC)
13C
1H
NMR Spectroscopy
Protein NMR
2D NOESY
NMR Spectroscopy
Protein NMR
2D NOESY
NMR Spectroscopy
Protein NMR
Isotopically labeled proteins
NMR Spectroscopy
Protein NMR
1H-15N
HSQC (protein’s fingerprint)
15N
1H
NMR Spectroscopy
Protein NMR
Signal overlap problem alleviated by 3D & 4D NMR
110 ppm
120 ppm
130 ppm
120 ppm
130 ppm
110 ppm
15N
15N
15N
1H
1H
1H
6.5 ppm
6.5 ppm
6.5 ppm
NMR Spectroscopy
Protein NMR
Signal overlap problem alleviated by 3D & 4D NMR
2D
110 ppm
3D
120 ppm
130 ppm
120 ppm
130 ppm
110 ppm
15N
15N
15N
1H
1H
1H
6.5 ppm
6.5 ppm
6.5 ppm
F2 (15N)
F3 (NH)
NMR Spectroscopy
Protein NMR
Signal overlap problem alleviated by 3D & 4D NMR
NMR Spectroscopy
Protein NMR
Signal overlap problem alleviated by 3D & 4D NMR
NMR Spectroscopy
Protein NMR
Assignment - Triple Resonance Experiments
H
C
H
N
C
C
H
H
O
3D HNCA
NMR Spectroscopy
Protein NMR
Assignment - Triple Resonance Experiments
NMR Spectroscopy
Protein NMR
Assignment - Triple Resonance Experiments
15N
ppm
115
118
122
125
116
130
128
40
45
13C
50
55
6.5
6.2
7.0
7.2
1H
ppm
7.8
8.5
8.0
ppm
NMR Spectroscopy
Protein NMR
Assignment - Triple Resonance Experiments
NMR Spectroscopy
Protein NMR
Assignment - Triple Resonance Experiments
HNCA
130 ppm
8.5
HN(CO)CA
130 ppm
8.5
i-1
HNCA
40
HN(CO)CA
125 ppm
125 ppm
HNCA
40
125 ppm
HN(CO)CA
125 ppm
40
45
45
45
50
50
50
55
55
55
7.5
7.5
i
7.5
7.5
i+1