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40-042 Analytische Chemie IV
Structure Determination by NMR
Prof. Bernhard Jaun
Office: ETH Hnggerberg HCI E317 Phone: 01 632 31 44 e-mail: jaun@org.chem.ethz.ch home page: http://www.jaun.chem.ethz.ch
Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV
Contents
1. Practical aspects of pulse Fourier transform NMR spectroscopy 1.1 1.2 1.3 1.4 1.5 1.6 1.7 2. The basic NMR experiment: physical description Excitation by RF pulses Digitization, window functions and fourier transform Quadrature detection Phase cycles and Z-gradients Dynamic range and solvent suppression The principal components of an NMR spectrometer
The principle of 2D-NMR 2.1 2.2 2.3 The basic idea A meaningless experiment Quadrature detection in 1
3.
Homonuclear shift correlation through scalar couplings 3.1 3.2 3.3 3.4 COSY. Determination of coupling constants from COSY cross peaks. TOCSY INADEQUATE: homonuclear 13C-13C double quantum spectroscopy.
4.
Spectral editing 4.1 4.2 4.3 Spin echo building blocks Heteronuclear J-modulation Polarisation transfer: INEPT and DEPT
5.
Heteronuclear shift correlation through one bond and long range couplings 5.1 5.2 5.3 5.4 Proton detected methods Proton detected H,X-COSY HSQC and HMQC HMBC
6.
Relaxation, dipole-dipole couplings and NOE 6.1 6.2 6.3 Longitudinal and transversal relaxation NOE in a two spin system without scalar coupling The mechanism of dipolar relaxation
7.
Homonuclear 1D-NOE difference spectroscopy 7.1 7.2 The steady state NOE in homonuclear multi-spin systems Practice of 1D-NOE difference spectroscopy
8.
Kinetic NOE-spectroscopy 8.1 8.2 NOESY NOE in the rotating frame: ROESY
9.
On the influence of dynamic processes on NOE spectra 9.1 Overall molecular tumbling, internal rotation (conformational changes) und chemical exchange 9.2 How to proceed with molecules with 0c near 1
10.
Combined application of several methods 10.1 Typical structure problems with organic molecules and suitable strategies
11.
References and additional reading 11.1 Textbooks 11.2 Homonuclear correlation through scalar coupling 11.3 Heteronucleare correlation through scalar coupling 11.4 NOE 11.5 Coupling constants und Karplus relationships 11.6 Abbreviations and Acronyms 11.7 On the presentation of 2D-NMR data in the experimental part of a thesis or publication.
What we would like to achieve with this course:

The student knows which method yields which type of structural information. The student is able to choose the most promising combination of methods for the solution of a given structural problem. Since spectrometer capacity is expensive, the selection of an optimal strategy depends on technical (feasibility) as well as on economical (fast and simple) factors. The student knows how to interpret each type of spectrum and how to extract the pertinent data. The student is aware of the possible artifacts and errors of interpretation for each method. The student knows the NMR vocabulary and is able to correctly describe NMR data in publications.
Subjects that are not part of this course:

Comprehensive treatment of the underlying theory (quantum mechanics of NMR) (-> courses by Prof. Schweiger and Meier). - Solid state NMR Practical "nuts and bolts" of carrying out the measurements on the spectrometer. (-> Praktikum Analytische Chemie) Application to large biopolymers such as proteins and nucleic acids (-> course by Prof. Wthrich) and calculation methods (simulated annealing, distance geometry etc.) used to derive solution structures of biopolymers from NMR data. Shorter oligopeptides, oligonucleotides und oligosaccharides, however, will be discussed as examples or problems.
Outline of the course:

Lectures on the individual methods with discussion of example spectra. Problems (real life spectra) for each of the discussed methods. Problems on structure elucidation of medium to complex organic molecules with combined methods (in the last ca. 1/3) of the course.
1.1
1.
Practical aspects of pulse Fourier transform NMR spectroscopy
1.1 The basic NMR experiment: physical description

Spin Component of the angular momentum of nuclei, electrons (and other elementary particles) that cannot be described as orbital momentum. Its origin is only understandable in terms of relativistic quantum mechanics. The magnetic moment The magnetic moment () associated with the orbital angular momentum (L) of a charged particle is given by:
L=rxp
e-
The spin angular momentum J is associated with a magnetic moment as well:
= J
, the gyromagnetic ratio is a fundamental property of each nuclear isotope with non-zero spin
Interaction between the magnetic moment und an external magnetic field Classical physics:
z
The torque T acts on . In response,
precesses around the direction of B 0 (analogous to a spinning top under the force of gravity) with the circular frequency 0 [rad/s], which is called the Larmor frequency.

B0
T = x B0
T x
0 = B
Epot = - . B0
1.2
Quantum mechanics: Quantum mechanical description of the spin angular momentum J : J = h I

: nuclear spin operator I
I : spin quantum number of the nucleus, a property of each isotope (I = 1/2. n; n=0,1,2).a) The z-component (parallel to the external field) of the spin angular momentum can only assume certain values governed by the magnetic quantum number mI : Jz = h mI mI = - I, - I + 1,..,0,, I - 1, I mI magnetic quantum number
This leads to 2I+1 allowed states. For nuclei with I = 1/2, which are of predominant interest in organic chemistry, only the two states with mI = -1/2 and mI = +1/2 are possible. The interaction energy for each state with a static external magnetic field along the z-axis is E = - z . B0 = - J z B0 E = - h mI B0 The energy difference between the two states is:
E = - h (1/2 - (- 1/2)) B = - h B0
0
In order to achieve resonance, the energy of the irradiated radio frequency has to match the energy difference between the two states:
E = h = h = - h B0
The Larmor (circular) frequency is:
i = - (1-i)B0z
With I = resonance frequency (rad/s) of spin i with shielding constant i The resonance frequency for a given isotope is proportional to the gyromagnetic ratio and to the external magnetic field.
a)
The spin quantum numbers of nuclei follow the rules: A = mass number Z = number of protons (nuclear charge) N = number of neutrons A even -> I = integer Z even, N even -> I=0 Z odd, N odd -> I=1,2,3... A odd -> I= 1/2, 3/2, 5/2 ...
A=Z+N
1.3
Macroscopic magnetization M Experimentally, only the total magnetization M of the sample inside the RF coil can be detected. corresponds to the vector sum of the magnetic moments of all spins. M=

sum over total sample volume inside the coil
For I = 1/2, > 0 (e.g. 1H):
B0 mI = + 1/2 z
B0 z
x mI = - 1/2
Because the magnetic moments are distributed statistically in the xy plane, there is no net transverse magnetization. In the Boltzmann equilibrium and for > 0, the population of the (mI = +1/2) state is slightly larger than that of the (mI = 1/2) state. This leads to a small residual magnetization in the direction of the external field B 0 . The energy difference between the two states (: mI = +1/2; : mI = 1/2) is very small. For 1H at 14.1 Tesla (600 MHz) the ratio of the two populations is only: N+1/2 / N-1/2 = 1.0002. Because 0 is proportional to and B 0 , nuclei with high are more sensitive than those with low spectrometers increases approximately according to B3/2.

and higher magnetic fields increase the sensitivity dramatically. In practice, the sensitivity of NMR
1.2 Excitation by radio frequency pulses

Rotating coordinate frame: The Larmor frequencies in modern NMR spectrometers are in the order of 30 - 900 MHz. On the other hand, the differences between the individual spins of the observed nucleus (chemical shifts and scalar couplings) are typically in the 1 Hz to 20 kHz range. In order to make the description of the dynamics of the magnetization both mathematically and visually easier, it is usual to use a coordinate frame which rotates around the B 0 = z-axis with the circular frequency 0.
1.4
The resulting "stroboscope" effect allows to describe the precession in terms of frequency differences = - 0. In the following, we will use the rotating frame for all vector diagrams. In modern NMR spectrometers with superconducting magnet coils, the magnetic field is parallel to the axis of the sample tube. The radiofrequency coil, which transmits the excitation pulses and the induced signal to and from the sample to transmitter and detector, respectively, is a saddle coil that generates and detects RF fields having their magnetic component B 1(t) orthogonal to the constant external field B0. The relative orientation of B1 vectors in the xy plane can be controlled by changing the relative phase of the irradiating RF. Irradiation of radiofrequency corresponding to the Larmor frequency of a given nucleus for a short time (an RF pulse of frequency = 0/2 and duration ) induces a complicated "spiral" movement of the
macroscopic magnetization M away from the z-axis towards the xy plane. In the rotating coordinate frame this process, which is called nutation, is a simple rotation of M around the axis of the field B 1. The nutation angle () is a function of both, the RF field strength B 1 and of the pulse duration (it is proportional to the integral of the RF pulse): = B 1 [rad]. In practical work, the amplitude of the RF field is usually given as B 1/2 [Hz]. It can be calculated if the duration necessary for a nutation of =90 is known: B 1/2 = 1/(4. (90)). Note: The spectrometer software uses parameters in the unit decibel (dB) attenuation from the maximal output in order to control the amplitude of RF pulses. Since these values are different for each instrument/amplifier/probe head combination, one should always use the absolute RF amplitude B1/2 [Hz] in publications.
M z0
B1
My0 2 -x x
1.5
Dependence of the excitation band width on the duration of the pulse Because the nutational angle is proportional to the integral of the RF pulse, the same nutation can be achieved either with a long weak pulse or with a short intense one. However, this holds only for spins, which resonate exactly at the frequency of the transmitter ( = 0). The bandwidth of excitation ( the frequency region in which spins are more or less equally excited) is directly dependent on the intensity of the pulse (peak to peak voltage, B1 amplitude). The first zero crossing of the excitation function occurs at 0/2 B1/2 Hz. Short intense pulses (so called hard pulses) are unselective and excite a broad region of the spectrum, long weak pulses (so called soft pulses) are selective and excite only a narrow region around the transmitter frequency. Continuous wave irradiation with very weak amplitude during 0.5-5s allows to irradiate a single line and is used for homodecoupling and presaturation of solvent signals.
Phase and amplitude of excitation
B1
Phase and amplitude of excitation
B1 Offset Effects Spins resonating at frequencies different from the transmitter frequency experience an effective RF field B1 eff that is the vector sum of B1 and of a component along B0: tan = 2(-o)/B1
Nutation around B 1 eff with (90) no longer follows a grand circle. For 90 pulses, the longer path and slightly stronger field B 1eff compensate each other. Therefore, 90pulses are much less sensitive to offset effects than 180 pulses. Pulse sequences are usually based on the assumption that offset effects are negligible. In reality, offset effects lead to artifacts and loss of signal in pulse sequences such as DEPT and heteronuclear shift
1.6
correlation, which depend on accurate 180 pulses. In order to minimize offset effects, high amplitude pulses for unselective excitation are standard in modern instruments. In practice, probe heads and amplifiers (typically 300W for X-nuclei) on a modern high resolution spectrometer can deliver 90 pulses as short as ca. 7 s (B 1/2 = 35 kHz). Higher power would lead to arcing in the probe and could destroy the probe head or amplifier. Practical example: 11.7 T (125 MHz for 13 C / 500 MHz for 1H): chemical shift range 13 C: -10 to 240 ppm = 15.6 kHz. Offset of a carbonyl signal: 13.5 kHz.-> With B1/2 = 35 kHz and transmitter frequency at 110 ppm -> = 21.
Offset effect z z
M B eff y x y M0
Beff
B1
tan = 2 ( - 0 ) / B 1 x
Evolution of magnetization in the xy plane after excitation by an RF pulse If equilibrium magnetization Mz is excited with an RF pulse, transverse magnetization (with components along x and y) is created. This corresponds to promotion of spins to the state and therefore to the generation of single quantum coherence (excitation of a mI = 1 transition). After the pulse, the magnetization in the xy plane evolves due to the chemical shift and the scalar coupling between spins as shown below:
1.7
Evolution of transverse magnetization under chemical shifts and scalar couplings
Mx = M y0sin(-Jt)
Mx = My0 sin(t)
My = M y0 cos(-Jt)
<0
y My = My0 cos(t) >0
My = My0 cos(Jt)
x Evolution of scalar coupling in the xy plane: shown is the A-part of an AX system with JAX > 0 and A = 0
x Evolution of chemical shifts in the xy plane
In the coil of the probe head, the precession of magnetization in the xy plane induces a very weak RF signal (V), the so called free induction decay (FID), which is amplified and recorded during the acquisition time t2 (typically 0.1s to 5 s). For practical reasons, the frequency of the transmitter and a so-called intermediate frequency are subtracted from the original signal such that the final signal entering the digitizer is in the kHz range (for details see 1.7). The FID decays with time due to the transverse (T 2) and longitudinal (T1) relaxation processes. T1 is the characteristic time for recovery of z-magnetization (return to Boltzmann equilibrium), whereas T2 is the characteristic time by which the coherence of transverse magnetization is lost because of dephasing of the individual spin vectors (for details see chapter 7.1). T2 is correlated with the line width of a signal in the NMR spectrum. The time domain signal (S(t), FID) is an interferogram of all frequencies corresponding to the individual lines of the NMR spectrum. The spectrum S() has to be calculated from the time domain signal by the mathematical operation of a Fourier transform. Since computers can only do discrete Fourier transforms, the analog time domain signal has to be converted into a series of discrete numbers S(t0 + t) equidistant in time by the analog to digital converter (ADC, digitizer).
1.8
1.3 Analog to digital conversion und Fourier transform

In order to allow reconstruction of a periodic signal such as a sine or cosine function from discrete data points, one has to digitize with at least twice the frequency of the periodic signal (Nyquist theorem). Therefore, the time interval between two data points, called dwell time (tdw ), has to be tdw 1/(2F) [s] when the signal most distant from the transmitter has a frequency 2(-o) = F . Because single channel detection cannot distinguish between positive and negative frequencies, the transmitter has to be set at one of the edges of the spectrum. Practical example: 1H at 500 MHz. Transmitter at 16 ppm, TMS at 0 ppm. F = 8000 Hz, t dw < 62.5 s. With quadrature detection (standard nowadays), the transmitter can be set in the center of the spectrum which reduces the spectral with by half: F' = F/2 and t'dw 1/(2F') = 1/(F). However, because two data points have to be collected for each time increment (the ADC alternately digitizes the signals of the two channels), the dwell time allowed for each channel is again 1/(2F). Example: 1H at 500 MHz. Transmitter at 8 ppm, TMS at 0 ppm. F =4000 Hz, tdw < 125 => 62.5 s per channel. With the availability of faster digitizers, modern high-end instruments allow to digitize much faster then dictated by the Nyquist theorem (so called oversampling). The redundant data points are used for digital filtering, giving much sharper cutoffs than with analog filters. Folding (Aliasing): Illustration of the Nyquist theorem:
1
0.5
-0.5
-1
10
Acquisition time (ms) Solid line: cos(2t) with =300 Hz; broken line: cos(2()t) with N = 1000 Hz, = 300 Hz. The dwell time is tdw =1ms, corresponding to a Nyquist frequency (2F) of 1000 Hz. Both signals give identical digitized data and the signal from the broken line would be folded into the spectrum after FT. Under these conditions, the highest correctly digitized frequency would be 500 Hz.
1.9
Signals that are outside the limits given by the Nyquist theorem (| o | > 1/(2t dw )) will be folded around the edges of the spectrum. Not only signals, but also noise is folded into the spectrum from regions outside the spectral width. This makes it necessary to use computer settable analog cutoff filters that are set to ca. (1.25*SW/2). For real FT, folding occurs around the nearer edge of the spectrum, for complex FT around the far edge ( see quadrature detection). Because no analog filters act in the artificial time domain t1 of 2D spectra, folding is of particular importance in the 1 dimension of 2D spectra.
Folding after a real Fourier transform (cos-transform)
signal with 1 < N
2N
(2)
folded
0 Hz
-N
Folding after a complex Fourier transform
signal with 1 < N
2N
0 Hz
(2 )
-N
folded
1.10
Fourier transform After single channel detection, the time domain array of data points is transformed by a discrete real FT (cos-transform). With quadrature detection the data in the two channels are used as the imaginary and real part of a complex FT. The algorithm used is FFT, which, using precompiled sine tables and coefficient swapping instead of multiplications is very fast on todays computers.
Complex analytical FT:

+
Complex discrete FT dt
s() =
{f (t) + if (t)}e
x y
it
S(i) = {SA (kt) + iSB(kt)} exp(i2lk / N)

k=0
N 1
Phase correction After the Fourier transform, the real and imaginary parts both contain the spectrum but with a phase difference of 90, in other words, orthogonal linear combinations of the absorption A and the dispersion spectrum D. During the process of zero order phase correction, a mixing coefficient is determined interactively such that the "real" part of the spectrum (the one displayed on the screen), contains the pure absorption spectrum. R = A cos + D sin A = R cos + I sin Frequency dependent phase errors are approximately corrected according to = 0 + c in the 1
st
order phase correction. Frequency dependent phase errors are typically the result of delayed acquisition after the end of the pulse sequence.
Digital resolution The acquisition time corresponds to the dwell time multiplied by the number of data points acquired in the time domain ( taq = tdw . n t2). After the Fourier transform, the number of data points of the real spectrum is half the acquired points (n2=nt2/ 2), evenly distributed over the spectral width (sw). Therefore, the digital resolution in the frequency domain is 2sw/nt2 [Hz/Pt]. Because t dw = 1/(2sw) the digital resolution is 1/taq. In order to correctly digitize a well-resolved spectrum with natural line widths of 0.2 Hz, one has to acquire for 10s (at least 2-3 data points per line).
1.11
Zero filling Extension of the measured time domain signal by an arbitrary number of zero data points before Fourier transformation leads to an interpolation of data points in the frequency spectrum. This gives smoother data but can not recover resolution that was lost by too short an acquisition. Since, in 2D experiments, the number of acquired points in the time domain t1 is directly proportional to the experiment time, zero filling by at least a factor of two is standard in the t1 / 1 dimension.
Convolution and window functions The natural envelope of a (strictly: single spin) FID is an exponential function according to: t / T2 I( t ) = Io e ite The effective line width 1/T2* is the sum of the natural line width 1/T2 broadening 1/T* (e.g. due to a non homogeneous magnetic field) 1/T2* = 1/T2 + 1/T* Fourier transformation of an exponential function gives a Lorentz function, the natural line shape of a single spin NMR signal. Multiplication of the time domain signal with another exponential function before the FT does not change the Lorentz nature of the frequency domain signal but changes the apparent line width: multiplication with e-t/a leads to line broadening concomitant with improved S/N whereas multiplication with e+t/a narrows the lines and drastically deteriorates the S/N. Multiplication of the time domain signal with a Gaussian ( e t
2 /a and
the instrumental line
) leads to Gaussian instead of
Lorentzian line shapes in the spectrum. Since Gaussians have a much more narrow base than Lorentz lines, this improves the apparent resolution of multiplets without serious costs in S/N. Stopping the acquisition before the analog signal has fully decayed into the noise is equivalent to multiplication of a step function into the FID. After the FT, this gives spectra with wiggles on both sides of each signal (the FT of a step function is a sinc (sin x / x) function). This can be avoided if the end of such an FID is multiplied with a half Gaussian function (apodization).
1.12
1.4 Quadrature detection
after (/2)
-x
y >0 y x fx (t)
sint
<0 x y - sint
fy (t) = cost
Simultaneous acquisition of the signal by two detectors that are 90 out of phase allows to distinguish between positive and negative frequencies. The signals from the two channels are combined as the real and imaginary part of the integrand in the FT.
+
s() =
{f (t) + if (t)}e
x y
it
dt
complex FT
1.13
Illustration of the principle:

cos(-t) = cos(t)
+ SW/2
0 Hz
- SW/2
sin(-t) = -sin(t)
+ SW/2
0 Hz
- SW/2
sum
+ SW/2
0 Hz
- SW/2
1.14
Redfield method of quadrature detection
transmitter frequency largest absolute frequency to be digitized single channel detection mirror images for all signals
+ SW
+ SW/2
- SW/2
- SW
dwell = 1/ (2 SW/2) = 1/ SW
SW* = 2 SW -> dwell = 1/ 2 SW
A shift of the receiver phase by 90 from one data point to the next one leads to an apparent shift of the spectrum by 1/(4SW*): the mirror images no longer overlap
+ SW
+ SW/2
- SW/2
- SW
After the FT, the two mirror images are folded on top of each other and the reference is shifted back by SW*/4 = SW/2
Redfield method: the digitizer rate is doubled: t dw = 1/(2 . SW) This gives the same number of data points as with true two channel detection but in a single file. The time domain signal is the integrand of a real (cos) Fourier transform:
+
s() =
f (t)e
x
it
dt
This method is also called TPPI (time proportional phase increments), in particular, if used in the t1 dimension of a 2D spectrum .
1.15
1.5 Phase cycles Cyclops

pulse # transmitter phase -x A: 1 -x -y y B B: x A + 2 FID channels A, B memory
+ 1
-x A: 2 -y -y x A y B 2 B: + 1
-x A: 3 x -y y B B: x A
1 2
-x A: 4 y -y y B 2 B: x A + 1
1 total: 2
A-B-A+B = 4 x cos t channels A and B are fully equalized B+A-B-A = 4 x sin t
1.16
Phase cycles are not only used to balance the contributions of the two detector channels but also in order to select desired coherence transfer pathways and to eliminate undesired contributions to the signal. This is possible because zero- and multi-quantum coherence respond differently to a phase shift than single quantum coherence. A phase shift of 90 leaves zero quantum coherence unshifted, shifts single quantum coherence by 90 and double quantum coherence by 180. That allows constructing phase cycles that lead to coherent addition of the desired signal but eliminate the undesired components by subtraction (a 180 phase shift on alternate scans is equivalent to a difference spectrum).
Disadvantage of phase cycles:

1. The undesired components are eliminated by subtraction. Therefore, the full signal, including the unwanted components, is entering the receiver and the receiver gain has to be set to accommodate the full signal. Good subtraction requires very high spectrometer stability over the length of the experiment; otherwise, non-perfect subtraction of large unwanted signals gives residual artifacts in the spectrum. 2. Suppression of unwanted coherence transfer pathways requires a certain minimal length of the phase cycle (e.g. 16 or 32 scans per FID). This imposes a lower limit on the experiment time even if the signal to noise ratio of a sample would allow recording the spectrum with only one or two scans per FID. Gradients volume sensed by the RF coil + A A B C D E 0 B B +B(z)
0 0
B C D E
The disadvantages of phase cycling mentioned above can be avoided if coherence transfer pathways are selected using z-gradients. A gradient coil in the probe head generates a linear field gradient along the z-axis that adds itself to the main field B0. If such a gradient of amplitude g (usually given in Gauss/cm) is applied for the time t g, the frequency of precession of xy-magnetization depends not only on and J but also on the location of the spin along the z-axis of the sample. If the gradient is strong
1.17
and long enough (it is the integral under the gradient pulse that counts), the frequencies of individual spins will be spread according to their z-coordinate and the net xy-magnetization is no longer detectable. Application of a gradient in the opposite direction with equal length and amplitude reverses the dephasing process and leads to a gradient echo when the spins in all volume elements reach the original phase coherence.
/2
echo acquisition
RF
gradients tg in phase dephased
tg refocussed
As with phase shifts, coherences of different order respond to gradients in a different way. Zero quantum coherence precesses with the difference of the frequencies, double quantum coherence with the sum. Accordingly, magnetization that was dephased by a gradient as single quantum coherence, and then transformed into double quantum coherence by a pulse, will not be refocused by a gradient of opposite sign and equal amplitude and length. Application of gradients at suitable places in the pulse sequence is therefore an alternative method for selection of desired coherence transfer pathways.
tg
tg = p Bz (t)dt
0
p = coherence order; 1 for SQC, 2 for DQC, 0 for ZQC; tg = dephasing angle
Advantages of gradients for coherence transfer pathway selection

Since the unwanted magnetization is not refocused, the receiver does not detect it at all. This allows setting the receiver gain according to the desired signals only and eliminates the necessity for subtraction and the artifacts related to it. Since phase cycling is no longer mandatory, samples with a good S/N ratio can be measured with only one or two scans per FID, which reduces the experiment time dramatically.
1.18
Disadvantages of gradients
Because the refocusing of magnetization that was dephased by a gradient depends on the zcoordinate of a given molecule to remain constant during the experiment, diffusion leads to a loss of refocusable signal. This imposes an upper limit on the duration of the pulse sequence. Gradient spectroscopy requires additional hardware: the probe head has to be equipped with a gradient coil and a gradient amplifier that can deliver stable and high currents (typically 10A) is needed. Because of the enormous advantage of using gradients, this equipment is now standard for high end spectrometers.
1.6 Dynamic range and solvent suppression
+volt Receiver most significant bit
least significant bit
ADC 12 Bit or 16 Bit
analog signal - volt
The receiver gain is set to give correct digitization of the highest voltage by the ADC.
Dynamic range = ratio between the strongest signal and the weakest signal to be digitized. Example: 1 mM protein in H2O; (110 M in protons) dynamic range = 110/0.001 = 105
1.19
Accumulation:
Modern instruments have 16Bit ADCs for high resolution work and 32 or 64 Bit computer words. This corresponds to a dynamic range of 32768 : 1. The signal is accumulated according to S(N) = N. S(1).Correctly digitized noise will accumulate as N= N1/2.N(1). Therefore, S/N improves with the square root of the number of accumulations. However, this requires that the noise be digitized correctly. Because, in samples of very high dynamic range, the receiver must be set to accommodate the largest signal, there is a risk that the smallest signals (including noise) are no longer correctly digitized because the corresponding voltage is below the least significant bit of the ADC. In this situation, accumulation does not improve the S/N. Solvent suppression If the molecules to be analyzed have exchangeable protons (NH, OH) and have to be measured in protic solvents such as water or methanol, the exchangeable protons are replaced by 2H of the deuterated solvent (D 2O or CD 3OD) and are no longer detectable. In particular with oligopeptides and oligonucleotides, the NH protons are very important for the structure analysis. Therefore, one usually measures the spectra in H2O / D2O 9:1 or in CD3OH, which makes it necessary to suppress the extremely intense solvent proton signal in order to be able to measure the analyte signals correctly. Depending on the exchange rate of the NH (OH) protons, two strategies are used:
/2
-x selective
x
-x selective
WATERGATE
2
RF
AQ
z gradients
If the exchange is relatively slow on the time scale of T1 (ca < 0.01 s-1), the solvent signal is saturated by a highly selective cw-irradiation of 1-5s duration at the beginning of the pulse sequence. This so called presaturation method can be used successfully for most amide NH in oligopeptides. The iminoand amino-NH protons in oligonucleotides, however, exchange too fast for this method: presaturation of the solvent signal leads to transferred saturation of all NH signals as well. In this situation, one has to use a method that does not excite the solvent signal, but all other signals as uniformly as possible. The corresponding methods include jump-return, WATERGATE and excitation sculpting. The last two methods use pulsed z-field gradients and are among the best water suppression techniques available today.
1.20
1.7 NMR spectrometer hardware Block diagram of a modern high resolution liquids NMR spectrometer
Network usually a UNIX ETHERNET Workstation
Host Computer RF-Unit

Acquisition Computer: Pulseprogrammer ADC Buffer memory
field-frequency stabilization Lock(2H)-RF channel

1
Magnet
Sample
H-RF channel main coil
X-RF channel Y-RF channel(Option)
Hard-Disks MO Disks Tape Output: Plotter Printer
Probehead Gradient unit (Option) Temperature controller Temperature sensor cooling gas N2 / air
Radio frequency unit
computer sum to memory real Dwell clock =0 Digitizer probehead =90 audio signal IF+ preamplifier imaginary
Receiver phase IF
+IF o o transmitter o pulse programmer
+ pin diode tuning matching
Synthesizer
1.21
Magnet and probe head
upper barrel pneumatic sample injection, ejection vacuum sample tube spinner He shim coils (RT) and Z0 supraconductor coil (B 0 ) and cryoshim coils
N2
isolated gas inlet (Dewar)
RT-shimcoils RF connectors cooling gas heater
Further reading T.D.W.Claridge, High Resolution NMR Techniques in Organic Chemistry, Pergamon, 1999, Chapters 2 and 3. J. Sanders, B. Hunter, "Modern NMR-Spectroscopy", Oxford University Press, 2nd Edition, 1992, chapter 1. H. Gnther, "NMR-Spektroskopie", Thieme, 3. Auflage, 1992. H. Gnther, "NMR spectroscopy : basic principles, concepts, and applications in chemistry^", 2 nd edition Wiley, 1996 H. Friebolin, "Ein- und zweidimensionale NMR-Spektroskopie", 3. Auflage, Wiley-VCH, 1999. F. K. Kneubhl, "Repetitorium der Physik", Teubner, 5. Auflage, 1994.
2.1
2. The principle of 2D-NMR spectroscopy

2.1 The basic idea
Preparation
Evolution
t1
Mixing
tm
Detection
t2
Acquire a series of FIDs:

t1
Starting with t1=0, the evolution time t1 is incremented by t1 (the "dwell time" in t1) from one FID to the next one.
t2 FT in t2 / 2
t1
A series of spectra in 2, aninterferogram("FID")in theorthogonaldirection(t1).

FT in t1 / 1
A 2D NMR spectrum: diagonal peaks (1=2) and cross peaks (1 2)

2.2
2.2 A meaningless experiment:

Labeling the t2-signal with chemical shifts during t1 COSY spectrum of an AX system without scalar coupling (JAX = 0)
t1
(/2)-x
t2 (/2) -x AQ z
y (/2) -x x x
t1
sin Xt1 sinA t1
z sin At 1 cosXt1
sin Xt1
z cosXt 1
X y X x cosAt1 A (/2) -x x A cosAt 1 y
The final amplitude of the signal depends on t 1 and on the chemical shift during the evolution time. In a 2D-experiment, t1 is incremented from FID to FID.
signal amplitude after the second pulse.
t1
2.3
Because there is no coherence transfer without coupling between A and X, all signals have the same frequency during t1 and t2 and the 2D spectrum contains only diagonal peaks. The actual acquisition during t2 is done with quadrature detection, which eliminates the mirror image. In the "artificial" t1 dimension, all signals will be mirrored around the 1 = 0 axis: Result:
In order to eliminate the mirror images in 1, the equivalent of quadrature detection has to be constructed in t1 As for 1D spectra, there are two methods: 1. Hypercomplex (Ruben, States, Haberkorn, RSH) 2. Redfield (Time Proportional Phase Increments, TPPI)
2.3 Quadrature detection in 1 2.3.1 The hypercomplex method (RSH) For each t1-value, two FIDs with a phase difference of 90 with regard to modulation in t 1 have to be acquired. This can be achieved by changing the phase of the 2nd pulse by 90.
2.4
y x t1 x
y (/2)-x x sin t1
y FID 1
y x t1 x
y (/2)-y x cos t1
y FID 2
For each t 1 value, FID1 and FID2 are stored in separate memory blocks. Together with the two quadrature signals from channels A and B in t2, one obtains four memory blocks. Complex FT in both, 1 and 2, gives a 2D spectrum that is a matrix of four blocks: rr, ri, ir, and ii. Phase correction in each direction mixes the two blocks in the corresponding dimension such that in the end, the rr block (the one that is usually displayed and plotted) contains the 2D spectrum with absorption line shapes in both dimensions.
quadrature detection in t2
quadrature detection in t1
FID R2 R1 I2 I1 FT(1)
FID2
FID 2
0
FID1
90
FID1
FT(2) complex
RI RR
II IR
90
complex
spectrum
2.3.2 TPPI Only one FID is acquired per t1 value, but the t1 increment is half of that used with RSH. This gives a single block with twice as many FIDs as in the hypercomplex mode. Each time t1 is incremented, the phase of the 1 st pulse is shifted by 90. The spectrum obtained after a real FT in t1 can be folded around 1=0 and the reference is shifted back into the middle of the 1-domain (see Redfield method).
2.5
Result:
-SW 1/2 0 +SW1/2 1
TPPI and RSH are equivalent with regard to experiment time, S/N and digital resolution. Certain artifacts, in particular so called axial peaks, appear at the center of the 1 domain for RSH but at the edge of the spectrum with TPPI. Folding in 1 is also different in the two cases (see folding with real and complex transforms).
3.1
3. Homonuclear shift correlation based on scalar coupling

3.1 COSY
AX AX system (1st order) with JAX > 0
SQ AX A1 ZQ X2 SQ DQ A2 X1
SQ AX A1
JAX A2 X1
JAX X2
SQ AX
A
Coherence: Coherence is a generalization of the concept of magnetization. In the quantum mechanical treatment coherence corresponds to off-diagonal matrix elements of the density matrix.
Single quantum coherence (SQC) corresponds to excitation of an allowed (mI = 1) transition and is
either equivalent to observable transverse magnetization or evolves into observable transverse magnetization under chemical shift and/or scalar coupling. In the example of an AX system given above, the transitions A1, A2, X1, X2 are SQCs.
Double quantum coherence (DQC) correlates two states differing by mI = 2 and connected by two
transitions originating at a common energy level of coupled spins (e.g. A1 and X2 via or A2X 1 via ).
Zero quantum coherence (ZQC) correlates two states differing by mI = 0 which are connected by two
transitions originating at an energy level common to two coupled spins (e.g. A 1 and X1 via or A2 and X 2 via ). In general, a system of p mutually coupled spins can develop coherence of order 0 to p (p-quantum coherence, multi quantum coherence = MQC). Coherences other than SQC (DQC, ZQC, MQC) are not directly observable. Starting from equilibrium z-magnetization, a single pulse can only generate SQC ( A 1, A2, X1 or X2). Further pulses acting on SQC of one of the coupling spins can generate SQC of the coupling partner(s) as well as DQC and SQC of the pair(s).
3.2
SQC(A1)
90 pulse non-selective
SQC(A1) SQC(A2) SQC(X1) SQC(X1)
DQC(A1X2) DQC(A2X1)
ZQC(A1X1) ZQC(A2X2)
This process, brought about by the second (mixing) pulse, is called coherence transfer. One way to illustrate the coherence transfer pathways in a pulse sequence is to use a system of horizontal lines as in musical notation.
/2
2 1 0 -1 -2 t1
/2
t2 Only SQC (usually -1) is detected during t2 In addition to SQC, the second pulse generates DQC and ZQC, which are not detectable. In order to obtain pure absorption lineshapes in both dimensions, the magnetisation that was SQC of order 1 and -1 during t1 has to pass the phase cycle.
2-spin system AX: COSY pulse sequence
3.1.1 Absolute value COSY (magnitude COSY) In the early days of 2D spectroscopy (COSY was the first 2D experiment proposed), quadrature detection in t1 was not yet implemented. In order to avoid mirror signals in 1, a phase cycle that selects either for -1 SQC or for +1 SQC in t1 was used (so called echo or anti-echo selection). This eliminates the quadrature images in 1 but leads to line shapes that are mixtures of dispersion and absorption (phase instead of amplitude modulation of the signal in t 1). In order to get reasonable contour plots of such 2D-spectra, one has to calculate the absolute value s = (r2+i2)1/2 of each data point (magnitude spectrum). The dispersive contributions to the line shape lead to long tails in both 1 and 2. When two such tails (star shaped ridges in the contour plot) cross each other, artificial cross peaks appear although no coupling exists between the two spins. The star-shaped cross- and diagonal peaks can be changed into box-shaped ones if a "pseudo-echo" (e.g. sin 2-function) window function is applied. However, this deteriorates the S/N and the fine structure of cross peaks is lost. Therefore, magnitude COSY has been replaced largely by methods giving pure absorptive line shapes such as DQF-COSY.
3.3
Magnitude COSY of an AX system (without pseudo echo filter)
Pseudo-Echo Filter
3.4
Resulting 2D-spectrum
t1-noise and symmetrization: Instabilities of the spectrometer and environmental influences such as temperature and pressure changes in the lab, acoustic noise from walking around or magnetic disturbances of street cars (Zurich Tram), elevators etc. lead to variations of the intensities of strong signals from one FID to the next one in addition to the (wanted) amplitude modulation. After the FT, this transforms into noise bands parallel to 1 at the chemical shifts (in 2) of strong signals. Because, in principle, a 2D COSY spectrum is symmetrical around the diagonal, whereas the t1-noise is not, so-called symmetrization can be used to eliminate t1-noise. The operation of symmetrization consists of setting the intensity values of the two points that are symmetric to the diagonal to the smaller of the two values. In practice, however, the two dimensions of 2D spectra are commonly measured with different digital resolutions (e.g. 2048 data points in t 2 but only 512 data points in t1). Even if identical numbers of points are used for FT in both dimensions (e.g. by zero filling in t 1), the resulting 2D spectrum is no longer truly symmetrical around the diagonal. While quite popular for a while (many examples in the literature), symmetrization is no longer used in modern practice.
3.5
3.1.2 Phase sensitive detection of COSY (PS-COSY) If COSY spectra are acquired with quadrature detection (RSH or TPPI) in t1, the cross peaks can be phased to give true antiphase absorption line shapes. However, the diagonal peaks are 90 out of phase with regard to cross peaks and therefore have dispersion line shape. The star-shaped form of the diagonal peaks often covers cross peaks close to the diagonal. In contrast to magnitude COSY, the fine structure of cross peaks is well resolved in PS-COSY and can be used to qualitatively judge the size of the coupling constants. For protons with more than one coupling, the active coupling (the coupling that causes the cross peak) can be identified, because the cross peak is in antiphase with regard to the active coupling, but in phase with regard to all other couplings (passive couplings)
1D-spectrum
cross peak antiphase in 1 and 2
+ +
X diagonal peak in-phase dispersive in 1 and 2 1
+ +
2 cross section at 1=X
JAX
3.6
PS-COSY of multi spin-systems: active and passive couplings Example: phase sensitive COSY of an AMX system with JAX = 0, JAM > 0, JMX > 0:
trace A
trace B
Double quantum filter A third 90 pulse, immediately after the 2nd one, reconverts some of the DQC generated by the second pulse back into observable SQC. Since DQC is twice as sensitive to phase shifts as SQC, it is possible to construct phase cycles that let only pass magnetization that was present as DQC between pulses 2 and 3. Because DQC is only possible for at least two mutually coupled spins, the double quantum filter eliminates all signals (including the diagonal peaks) of singlets. Furthermore, the cross peaks and the diagonal peaks can be phased into pure absorption in both dimensions in DQF-COSY spectra. This gives a much narrower diagonal and allows seeing cross peaks very close to the diagonal. Although the sensitivity of DQF-COSY is only half of that of magnitude or PS-COSY, the advantages predominate and make DQF-COSY the method of choice in today's practice.
3.7
/2
2 1 0 -1 -2
t1
/2 /2
t2
The 2nd pulse generates DQC (and ZQC), the 3rd pulse converts it partially back into SQC. The phase cycle or gradients select magnetisation that was present as DQC during .
2-spin system AX: DQF-COSY
Multiquantum filters In analogy to the double quantum filter, phase cycles that act as n-quantum filters can be constructed. They only let pass magnetization that was present as n-quantum coherence during . n-Quantum coherence requires n mutually coupled spins (each of the n spins couples with all others). In practice, systems with more than three mutually coupled spins are rare. In addition, the sensitivity of the experiments rapidly diminishes with higher coherence order n of the filter. A triple quantum filter, e.g., may allow distinguishing between the following three-spin systems:
HA
HB
HA
HB
HC
HC JAB, J AC , JBC all 0 JAC often = 0
cross peaks in TQF-COSY
no cross peaks in TQF-COSY
Prof. Dr. B. Jaun: Structure determination by NMR/Analytische Chemie IV
Examples for COSY variants
Menthol
CH3
OH
H3 C
CH3
Menthol in acetone-d6 300 MHz magnitude COSY processed with cos2-Filter
ppm
1.0
2.0
3.0
4.0 4.0 3.0 2.0 1.0 ppm
Menthol in acetone-d6 300 MHz magnitude COSY processed with pseudo-echo filter ppm
1.0
2.0
3.0
4.0 4.0 3.0 2.0 1.0 ppm
Menthol in acetone-d6 300 MHz PS-COSY
ppm
1.0
2.0
3.0
4.0 4.0 3.0 2.0 1.0 ppm
Menthol in acetone-d6 300 MHz DQF-COSY
ppm
1.0
2.0
3.0
4.0 4.0 Menthol in acetone-d6 300 MHz DQF-COSY expansion 3.0 2.0 1.0 ppm
ppm
0.8
0.9
1.0
1.1
1.2 2.4 2.3 2.2 2.1 2.0 1.9 ppm
3.8
3.2 Determination of 1H- 1H coupling constants from COSY spectra

1
H- 1H three-bond (vicinal) coupling constants depend on the dihedral angle around the central bond
according to the Karplus relationship. If suitable parameters for the Karplus equation are known, possible dihedral angles (in general, there are several solutions for a given 3 JHH ) can be deduced from accurately measured coupling constants. If the signals are well dispersed, the J-values can be determined with the highest precision from 1D spectra. If systems of higher order occur, the chemical shifts and coupling constants can be determined by iterative fitting with a simulation program (e.g. SWAN-MR, MESTRE-C, VNMR, g-NMR, NMRSIM). However, if the signals overlap in the 1D spectrum, one has to extract the coupling constants from the fine structure of cross peaks in appropriate 2D spectra.
Qualitative (large, medium, small) coupling constants can be estimated based on the antiphase / in
phase properties and the intensities of cross peaks in DQF-COSY spectra. The antiphase nature of the cross peaks with regard to the active coupling leads to a new, "antiphase Pascal triangle" for the line intensities in multiplets of protons coupling with several magnetically equivalent spins. In contrast to in phase spectra, central lines of multiplets with an odd number of lines can actually disappear. In an antiphase triplet, e.g., only the two outer sub-lines are seen in antiphase, whereas the central line vanishes. The distance between the two visible lines is twice the coupling constant. 1 1 1 1 1 2 1 0 etc. The precise determination of numerical J-values from antiphase cross peaks in DQF-COSY spectra is difficult for the following reasons: 1. The digital resolution in the 2 dimension of the 2D spectrum is usually much lower than in the 1H1D spectrum (and even worse in the 1 dimension). 2. Lorentz lines in antiphase partially cancel each other, if the linewidth is comparable to the splitting. The distance between the maxima of such strongly attenuated residual lines is larger than the coupling constant. In-phase lines with splittings not much larger than the linewidth are not baseline resolved and will have their maxima closer than the coupling constant. Thus, active coupling 0 -1 -2 -1 -1 -1 -1
constants will be overestimated and passive coupling constants will be underestimated.
3.9
1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 -5 -4 -3 -2 -1 0 1 2 3 4 5
1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 -5 -4 -3 -2 -1 0 1 2 3 4 5
1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 -5 -4 -3 -2 -1 0 1 2 3 4 5
/ LW = 4
/ LW = 3
/ LW = 2
1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 -5 -4 -3 -2 -1 0 1 2 3 4 5
1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 -5 -4 -3 -2 -1 0 1 2 3 4 5
1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 -5 -4 -3 -2 -1 0 1 2 3 4 5
/LW = 1
/LW = .5
/ LW = .25
in Phase
1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 -5 -4 -3 -2 -1 0 1 2 3 4 5
2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 -5 -4 -3 -2 -1 0 1 2 3 4 5
/LW = 0.1
/ LW = .1
3.10
E.COSY The best way to determine coupling constants from cross peaks is to measure the distance between two in-phase sub-lines that are not affected by antiphase cancellations. E.COSY is one method that produces cross peaks with only half of the sub-lines present in DQF-COSY. E-COSY is a weighted linear combination of nQF-COSY spectra with n = 2,3,4i (coherence order of the nQ-filter, in practice, one stops the series at n=3 or 4). The cross peaks in nQF-spectra with different n have different symmetry properties. Therefore, summation cancels half of the sub-lines. The measurement of true E.COSY spectra with good S/N is very time consuming. An alternate pulse sequence that gives E.COSY type cross peaks but with improved sensitivity is PE.COSY. E.COSY of an AMX 3-spin system
TQF-COSY DQF-COSY
E.COSY
The basic element of an E.COSY cross peak is an antiphase square which represents the active coupling. Each spin that passively couples with one or both of the actively coupled nuclei generates a displaced set of squares. The displacement vectors connecting identical corners of the squares have
3.11
the passive couplings as their projections in 1 and 2. Because only half of the sub-lines are present, the measurement of passive coupling constants via displacement vectors is quite accurate.
Analysis of E.COSY-cross peaks: displacement vectors.
JAB active JAC passive JBC passive JAD passive JBD passive
HA
HA
HB
HC 2 A/B cross peak JD(AB) JC(AB)
HD
HB
Procedure: draw the displacement vectors such that they connect the same corner of two squares and
make an angle between 0 and 180 with the horizontal axis. Each displacement vector represents one additional spin that is passively coupled to either A, B or both. The 2- and 1-projections of the displacement vector correspond to the passive coupling constants of this spin with H A and HB, respectively.
J C(AB) JAC D J D(AB) JBD JAD JBC C
The angle between the displacement vector and the 2-axis indicates the relative sign of the two passive coupling constants. If < 90, they have the same sign, if it is >90, they have opposite signs. In the example above, C > 90 and D > 90. For both vectors, one coupling constant is vicinal (usually positive) the other one geminal (usually negative). Therefore, JAC .JAD < 0 and JBC .JBD < 0.
3.12
SOFT-COSY as an alternative to E.COSY E.COSY requires the acquisition of several nQF-COSY spectra with high digital resolution in both dimensions. Since the number of points in t1 is directly proportional to the total experiment time, good E.COSY spectra take a lot of time (and disk space!). Often, it is a better strategy to acquire a series of SOFT-COSY spectra for the cross peaks of interest. SOFT COSY requires the use of selective excitation pulses; usually Gaussian shaped weak pulses of several milliseconds duration. Since in both, 1 and 2, only a narrow band centered on the chemical shifts of the cross peak is measured, high digital resolution can be achieved with relatively few data points in each dimension. The necessary simultaneous gauss pulse at two frequencies can be realized by a phase modulation of the pulse shape. SOFT-COSY cross peaks have the same structure than E.COSY cross peaks and may be analyzed in the same way using displacement vectors.
A B C the final 2D spectrum shows only this area C
the first Gaussian pulse excites only this region
the mixing pulse is a simultaneous 90 Gaussian pulse at both frequencies A
during t2, only this region is detected
Selective excitation by Gaussian pulses
t1 preparation evolution mixing
AQ
t2
3.13
3.3 TOCSY (or HOHAHA)
Isotropic mixing: In 1st order coupled spin systems, each line (transition) can be attributed to one of the
spins. In contrast, all coupled spins contribute to each line in higher order systems with 10 J. Through application of a strong B1 field that is collinear with the transverse magnetization, (a so called
spin lock) during the mixing time tm, the precession due to the chemical shifts is suppressed. In the
absence of chemical shifts, all coupled systems are high order systems during the time of the spin lock and in phase SQC is transferred from one coupled spin to the other. If the mixing time is long enough (typically 65-100 ms) transfer not only occurs to directly coupled spins, but to all spins in the same uninterrupted sequence of coupled spins. In the ideal TOCSY spectrum, each spin in the spin system gives a cross peak with all other spins in the system. In practice, the spin lock is implemented with composite pulse decoupling sequences such as MLEV17, DIPSY2 or FLOPSY. In these decoupling sequences, the CW pulse is phase modulated according to a specific pattern with cyclic permutation. Composite pulse spin locks have a much broader uniform excitation profile than a single phase CW irradiation of the same power. TOCSY pulse sequence:
/2
relaxation delay t1 MLEV17 or DIPSY2 or FLOPSY t2
In order to have an efficient spin lock for the full range of chemical shifts, the spin lock field should be near B 1/2 = 10 kHz. Such a strong B1 field for up to 100 ms duration is near the limit of what amplifier and probehead can safely deliver. In contrast to COSY, where antiphase SQC is transferred, net in-phase SQC is transferred in TOCSY. Therefore, all cross peaks have the same phase as the diagonal. The lineshape is nearly pure absorption for larger spin systems but can contain dispersive components, in particular for small spin systems such as an isolated AB system. The rate of transfer during the isotropic mixing is dependent on the coupling constants: the larger J, the faster the process. Small coupling constants in a chain of protons can act as a bottleneck and prevent full transfer from one end of the chain to the other. The pulse sequence of TOCSY is identical to that used in ROESY (the only difference being the stronger spin lock field used for TOCSY). In particular with large molecules, where the build up of
3.14
ROESY peaks is fast, the TOCSY spectrum can contain ROESY cross peaks as artifacts. Because the phase of ROESY peaks is opposite to the phase of the diagonal, simultaneous ROESY and TOCSY transfer between a pair of protons may even lead to the disappearance of certain cross peaks. Variants of TOCSY, which avoid some of these problems, are z-filtered TOCSY (elimination of the dispersive lineshape components) and clean-TOCSY (elimination of ROESY contributions).
TOCSY spectra with long mixing times (up to 100 ms) complement the information gained from DQFCOSY and are useful in cases of strong overlap. Typical examples are molecules with repetitive substructures such as oligosaccharides, nucleic acids and peptides: in all these molecules, each subunit has at least one proton that is well isolated in the spectrum: anomeric protons for oligosaccharides and nucleic acids, - or NH-protons for peptides. Since each residue is an isolated spin system, the TOCSY trace at the chemical shift of the wellisolated protons will ideally show all other protons belonging to the same residue.
Prof. B. Jaun: Structure determination by NMR/Analytishe Chemie IV
TOCSY examples
Examples TOCSY
Menthol in acetone-d6 300 MHz TOCSY with 84.4 ms mixing time
ppm
1.0
2.0
3.0
4.0 4.0 3.0 2.0 1.0 ppm
Menthol in acetone-d6 300 MHz TOCSY with 20.9 ms mixing time
ppm
1.0
2.0
3.0
4.0 4.0 3.0 2.0 1.0 ppm
3.15
3.4 INADEQUATE: homonuclear 13 C double quantum spectroscopy The central information about an organic molecule is the constitution of its carbon skeleton. Therefore, any NMR method that directly reveals the C-C connectivity would be extremely useful.
13
C- 13 C
coupling constants over one bond are substantial but strongly dependent on the hybridization of the bound carbons (typically 35Hz for sp3-sp3, 55Hz for sp2-sp2). The major problem, at least for unlabeled compounds, is the low natural abundance of 13 C: for a given carbon, the probability for
13
C is 0.011; for a pair of carbons to be

1
13
C- 13 C, it is only 0.00012. A
13
C-
detected method for correlation of carbon chemical shifts through JCC is 100 times less sensitive than 1D-13 C-NMR. This restricts the usefulness of such methods to samples giving S/N 10 in the broadband decoupled 13 C spectrum with a single scan. INADEQUATE pulse sequence:
90
180
90
270
t2 t1 ACQ
13C
= 1/(4JCC)
antiphaseSQCis generatedand isrefocussed
precessionofDQC
SQC
broadbanddecoupling
1H
INADEQUATE is a method using in phase

13
13
C- 13 C-double quantum coherence based on 1JCC . After excitation to
C-SQC by the first pulse, antiphase 13 C-SQC is allowed to develop while the 180 pulse in
the middle of the preparation period refocuses the chemical shifts. The third pulse converts part of the antiphase
13
C-SQC into DQC that precesses with the sum of the chemical shifts of the coupled
carbons during t1. It is reconverted into 13 C-SQC by the last pulse for detection during t2. Because DQC precesses with the sum of the chemical shifts of the two coupled carbons, the spectral width in 1 has to be twice as large as in 2. In order to save experiment time (proportional to the number of points in t 1), one often deliberately allows folding in 1 by setting identical SW in both dimensions. The spectra are usually displayed and plotted in magnitude or power mode.
3.16
- SW
A
B
Crosspeaksappearasapair ofdoubletssymmetricaltothe diagonal1=22. In2,eachdoubletisfoundat thechemicalshiftofthe correspondingcarbon. In1bothdoubletsappearat thesumofthechemicalshifts (1() = A+B,relativeto thetransmitter1 =0).
B 1
A
A+B
SW/2
+SW +SW/2
Disadvantages of INADEQUATE (besides its extremely low sensitivity):

INADEQUATE is a
13
C-detected method. Therefore, full relaxation of
13
C has to be allowed between
two scans. In particular with quaternary carbons, which can have relaxation times up to minutes, this would require unrealistically long experiment times. The large variation of 1JCC with hybridization (by more than a factor of two) makes it impossible to choose the delay optimally for all carbons, if the molecule has saturated and unsaturated carboncarbon bonds. In order to reveal all correlations, one may have to acquire two spectra with different values. INADEQUATE can be very powerful in biosynthetic studies when doubly labeled precursor molecules are fed to the organisms (so called bond labeling, e.g. with [1,2-13 C2]-acetate, usually diluted 1:10 with unlabeled precursor). As long as the bound pairs of carbons are staying together in all biosynthetic steps, INADEQUATE will be as sensitive as 1D-13 C-NMR and directly indicate which bonds have been preserved in biosynthesis and which of the labeled bonds have been broken.
3.17
INADEQUATE: a virtual example of interpretation
HO Cl
1
CH2OH OH
2
5 4
Cl
OH
- SW/2 folded signal in 1
folded once in 1
+SW/2 - SW/2 2 +SW/2
INADEQUATE example
INADEQUATE example
Menthol in acetone-d6 125 MHz 13C INADEQUATE [Menthol] = 2.3 M experiment time 12 h folded once in 1
80
70
60
50
40
30
20
ppm ppm
-20
20
40 40 20 0 -20 ppm
4.1
4. Spectral editing
4.1 Spin echo Spin echo building blocks (-180-) refocus precession due to chemical shifts and/or scalar coupling at a given moment in the pulse sequence. In heteronuclear spectroscopy, they can be used to eliminate the effects of chemical shift of either nucleus, of the heteronuclear scalar coupling or both.
Homonuclear two spin system without scalar coupling A und X y A y 180 -x A y y
X x x x Result: chemical shifts are refocused after the second delay Heteronuclear AX system (e.g. CH). The X-magnetization is shown. The 180 refocusing pulse acts only on X A= A= y A= y A= y 180 -x onX
x x x Result: X-chemical shift and heteronuclear coupling are refocused for X
Heteronuclear AX system (e.g. CH). The X-magnetization is shown. 180 refocusing pulses act simultaneously on A and on X: The A= and A=vectorsareexchanged. A= A= A= A= A= y y y 180 -x onA andX y
x x x Result: chemical shifts are refocused, but the heteronuclear coupling is not.
A=
Homonuclear AX system. A hard 180 refocusing pulse acts always on A and on X: The and vectorsareexchangedforAandX. X= X= A= X= X= y
y A= A=
y A= 180 -x A=
A= x
x x x Result: chemical shifts are refocused, but homonuclear couplings are not.
4.2
4.2 Heteronuclear J-modulation Editing of 13 C-spectra through heteronuclear J-modulation: APT

H3 C H2 C 2Jt Jt y +Jt
x x
HC Jt y +Jt
x
3Jt
y +2Jt
+3Jt
= 1/J
CH
= 1/J
CH
= 1/J
CH
= 1/J
CH
start of BB-{1H}-decoupling
180 -x(13C)
Chemical shifts are refocused. Decoupling prevents the multiplet sub-lines of separating again
= 1/J
CH
acquisition
CH3
CH 2
CH
Pulse sequence of ATP:

90 180
1/(2.1JCH)
13 C
1/(2.1JCH) acq
1H
broadband decoupling
4.3
The resulting ATP spectrum exhibits the same signals as a BB-{1H}-decoupled
13
C spectrum but the
phase depends on the number of attached protons: C and CH2 opposite to CH and CH 3. In contrast to DEPT and INEPT (see below), no polarization transfer is involved and quaternary carbons are detected. The sensitivity is actually lower than for the normal for nearly complete
13 13
C-spectrum, because one has to wait
C-relaxation between transients. Nowadays, APT has been replaced by DEPT
as the method of choice for the determination of the number of attached hydrogens. J-resolved 2D spectroscopy: heteronuclear J-modulation during t1. J-modulation is the basis of a 2D-experiment, which correlates heteronuclear one bond coupling constants with
13
C chemical shifts. Instead = 1/4J, the delay is incremented as evolution time t1.
13
The simultaneous 180 pulses on 1H and 13 C refocus the chemical shifts but not the heteronuclear coupling. After Fourier transformation, the 2D spectrum shows coupling constants in 1 and
1
chemical shifts in 2. J-resolved heteronuclear 2D is a good method for the accurate measurement of JCH coupling constants, but it has a low sensitivity (the basic sensitivity is similar to ATP but each
carbon signal is split into a multiplet). In today's practice, it has been replaced by the much more sensitive 1H-detected methods such as HSQC without 13 C decoupling (see chapter 5). Pulse sequence for heteronuclear J-resolved 2D spectroscopy:
90
t
13 C 1
180
t
1
180
acq
BB-dec
1H
2D-J-Resolved Spectrum:
JCH
4.4
4.3 Polarization transfer The gyromagnetic ratio of 1H is ca. 4 times as large as that of times higher (linearized Boltzmann equation) for 1H than for
13
C and ca. 10 times that of
15
N. At
Boltzmann equilibrium, the population difference between the two states is therefore roughly four
13
C. Polarization transfer steps in
heteronuclear pulse sequences transfer the higher population difference ("spin polarization") of protons to the X-nucleus which - in theory - increases the sensitivity of X-spectra by H/X. Principle: illustration for a CH-group
AX-system (A= 1H; X=13C, A/X=4) Heteronuclear coupling 1J CH A-part Approximate analysis of populations (linearized Boltzmann equation) Population differences at equilibrium 1H : 2 A ; 13C: 2 X; lets assume A = 4, X = 1 At Boltzmann equilibrium: A X -5 -3 -A-X -A+X A
1
A2
A1
X2
X1
X-part
after inversion of A1 X1 A X +A-X -A+X 3 -3
A2 3 5 +A-X +A+X A X X2 AX -A-X +A+X -5 5
Consequences of the inversion of A1: 13C-spectrum without broadband decoupling after a

13C-90 read pulse:
X 1X 2
X1 X 2 6
-2 -2
10
4.5
With natural 13C-magnetization eliminated by phase cycling:

8 00
-8
INEPT Illustrated for an AX system (shown is the A-magnetization)
90 -x
y x x y
1 4J
z X= 45 X= x y
180 -x(A) 180 -x(X)

45 x X=
X= y
1 4J
z X= y x X= x
z X=
90 y
y
X= one A-transition selectively inverted !
4.6
INEPT pulse sequence:
90-x 1
1H
180 -x 1 4J 180 -x 4J
90-y
90-x
INEPT
13 C
In this simplest form, INEPT gives antiphase multiplets for each CHn. For 1H-decoupling during acquisition, the antiphase multiplets have to be refocused before the start of acquisition and decoupling. The delay can be used to edit the resulting spectra for CH, CH2 or CH3: =1/(2J) gives only CH, =3/(4J) gives CH, CH3 positive, CH2 negative. Pulse sequence for refocused INEPT:
90 -x
1H
1 4J
180 -x
1 4J
90-y BB-dec 90-x 2 180 -x 2
180 -x
13 C
Unfortunately, the result is very dependent on the right estimate for 1JCH . Because one bond CH coupling constants vary dramatically (aliphatic CH n ca.. 125 Hz; CHn-O ca.. 140 Hz; HC= and HnC(O) 2 ca. 160 Hz, HC ca 200 Hz; HCCl3 220 Hz), it is normally not possible to choose a single value for the delay that matches the 1JCH for all carbons in a molecule. DEPT (see below) is much less sensitive to the correct match of the delay and is therefore today's choice for multiplicity editing of 13 C-spectra.
DEPT In contrast to the INEPT sequence, DEPT produces heteronuclear multiquantum coherence after the polarization transfer. Since a vector diagram can no longer represent this correctly, we just show the pulse sequence and describe the results.
4.7
DEPT pulse sequence:

(/2)
-x
()
-x
() editing pulse
-y
1/(2J)
1H
1/(2J) (/2)
1/(2J)
BB-dec
13 C
AQ
heteronuclear multiquantum coherence
In DEPT, the multiplicity editing is not done via a delay but through the pulse . DEPT is much less dependent on the correct choice of than INEPT. Even more tolerant is the variant DEPT-GL.
DEPT: dependence of phase and intensity of the signals on for different CHn:
+
CH
CH 3
0 CH
0 45 90 135 180
For the determination of the number of attached protons it is sufficient to take two DEPT spectra, one with =90 (only CH, positive) and one with =135 (CH and CH3 positive, CH2 negative).
Disadvantages of DEPT: a) Broad signals (short T2) are often not detected because DEPT is a
relatively long pulse sequence and all magnetization is lost before the acquisition starts. b) Signals of
13CH-groups of type aldehyde or imine (HC(R)=X) such as found in many heterocycles, are often not
detected in DEPT. The reason is twofold: the coupling constants are very large (ca. 200 Hz, whereas the delay is usually matched to an average coupling constant of 130-140Hz), and the chemical shift of such groups is at the edge of the 13 C-spectrum giving large offset effects.
5.1
5.
Heteronuclear shift correlation
5.3 1 H-detected Methods (also called "inverse" or "reverse" methods) On the relative sensitivity of NMR experiments The signal to noise ratio of NMR signals is proportional to:
(excited) (detected)3/2 B03/2 (number of transients)1/2 As discussed in chapter 4, polarization transfer from 1H (excited) to X (detected) leads to a gain in sensitivity of up to H/X. The gain in sensitivity (see table below) is even larger for methods in which protons are not only the excited nucleus, but also the detected one.
Relative sensitivity at 100% abundance without NOE Combination of nuclei

1H/31P 1H/13C 1H/15N
X excited X detected 1/10 1/32 1/300 Inv. gated 13C
H excited X detected 1/4 1/8 1/30 DEPT, INEPT
X excited H detected 2/5 1/4 1/10 PH-COSY
H excited H detected 1 1 1 HSQC, HMQC
Examples
Relative experiment times for identical S/N Combination of nuclei

1H/31P 1H/13C 1H/15N
X excited X detected 100 1024 100000
H excited X detected 16 64 1000
X excited H detected 6.25 16 100
H excited H detected 1 1 1
The relevant relaxation time for 1H-excited methods is T 1 of protons, which is usually much shorter than T1 of X-nuclei. Hence, 1H-excitation has the additional advantage that more transients can be acquired per time because only relaxation of protons has to be awaited in between transients.
5.2
The principle of proton detected 1H- 13C-shift correlation:

1
H-magnetization is excited and transferred to 13 C in the preparation period. The signal is labeled with C-chemical shifts during the evolution period t 1 and then transferred back to 1H SQC for the detection
13
period t2. Because only 1.1% of the protons are bound to

13
C, the 100 times stronger signal of the
12
C bound
protons has to be suppressed. If the selection is based on phase cycles, very high spectrometer stability is required; whereas with gradient based selection only the signal of reaches the receiver. Of the two coils of a heteronuclear probehead, the inner one, which is closer to the sample and therefore more sensitive, is usually tuned to the detected frequency. For optimal X-detection, the inner coil is tuned to X and the outer coil is used for 1H-decoupling. For 1H-detected heteronuclear experiments, the opposite arrangement is used: 1H on the inner coil and X on the outer coil.
13
C-bound protons
5.2 Proton detected H,X-COSY for abundant X nuclei

This 1H-detected method is convenient for the correlation of protons with abundant X-nuclei such as
31
P. The pulse sequence is the direct heteronuclear analog of COSY with two simultaneous 90 pulses
on 1H and X acting as the mixing pulse. At the beginning of the sequence, all natural 1H-magnetization is destroyed by a train of 180 pulses on 1H. Therefore, only the chemical shift and coupling of X evolve during the evolution time t1. The resulting 2D spectrum shows cross peaks in antiphase absorption in both dimensions.
180 90
1H
t2 n presaturation of natural 1 Hmagnetization
90 t1
90
31 P
Example: H,P-COSY allows to correlate sequential sugar units in oligonucleotides through two- and
three-bond couplings between the sugar protons and the
31
P of the phospho-diester linkage
CHOPO2O-CH-. Each phosphorous shows cross peaks to the 3'-H of the preceding and the 5'-H2 of the following sugar in the sequence.
5.3
5.3 HMQC and HSQC: one bond 1H-13C-correlation

1. HMQC (Heteronuclear correlation through Multiple Quantum Coherence)
exchanges DQC and ZQC refocuses J CH in t1 90 1 2JCH

1H
180 t
1
1 2JCH t2
90
heteronuclear DQC and ZQC
heteronuclear ZQC and DQC
90
GARP
13C-BB
13 C
dec
1H magnetization in antiphase with regard to J CH
The first 90 1H-pulse generates 1H-SQC; the first 90
13
C-pulse transforms this into heteronuclear
double and zero quantum coherence. The 1H-180 pulse in the middle of t1 refocuses the 1JCH coupling and swaps ZQC (evolution with C - H) and DQC (evolution with C + H). At the end of t1, the effects of 1H chemical shifts during the two halves of t1 cancel, and only C labeling remains. Homonuclear 1H- 1H-couplings evolve during t 1 as well as t2. The two halves of the 2D-TPPI spectrum before folding are symmetric with regard to the chemical shifts but not symmetric with regard to the tilt of each cross peak caused by homonuclear coupling. After Folding this leads to a spread (broadening) of the cross peaks in the 1 dimension.
JHH JHH
antiecho
JHH
JHH
JHH JHH
echo
after folding (TPPI)
in reality, because of limited resolution in 1
5.4
GARP-broadband 13C-decoupling during t2 serves to collapse the cross peaks which are otherwise split by the heteronuclear coupling 1JCH of 125-200 Hz (remember, it is the 13 C-satellites in the proton spectrum which are actually measured). Since broadband decoupling of all 1H-bearing carbons (ca 160 ppm) is demanding on the decoupler and heats the sample, in particular if it has a high dielectric constant, GARP decoupling during acquisition may be left out at the cost of a more complicated spectrum. 2. HSQC (Heteronuclear Single Quantum Correlation)
90 x
1 4JCH
1H
180 x
1 4JCH
90 y
t
1
180
t
1
90 y
1 4JCH 2
180 x
1 4JCH t
2
180
90
90
180
GARP
13 C
The preparation part of this sequence is identical to INEPT. During t 1,
13
C-magnetization precesses in
x,y while 1H-magnetization is in z. Therefore, neither chemical shifts nor homonuclear couplings of protons evolve during t1. The 180 pulse in the center of t 1 refocuses 1JCH . At the end of the evolution period, coherence is partially retransferred to 1H-SQC, which is detected (reverse polarization transfer). In contrast to HMQC, homonuclear couplings do not split the HSQC signals in 1. This leads to a clean absorption line shape in both dimensions and makes HSQC the better method in cases where high resolution in 1 is necessary. However, the HSQC pulse sequence is more complex than that of HMQC and therefore more dependent on correct pulse calibrations.
5.4. HMBC (Heteronuclear correlation through multiple bonds; long range variant of HMQC)
1
H- 13 C-scalar couplings over two and three bonds are in the 0 to 10 Hz range, comparable to 1H- 1H
couplings. Couplings over more than three bonds are usually very small and are rarely detected by HMBC. Unfortunately, the 2-bond and 3-bond coupling constants cover the same range and can not be distinguished by HMBC. Although less sensitive than HSQC and HMQC, HMBC is more sensitive than INADEQUATE by several orders of magnitude and delivers the same type of information: connectivity of the carbon skeleton including quaternary carbons. In contrast to INADEQUATE, HMBC also correlates fragments across heteroatoms.
5.5
90
1 2 1J
1H
CH
180
1 2 JCH
n
t1 2
t1 2 t
2
90
90
90
13 C
low pass filter: eliminates signals of directly bound protons

The following modifications are made to the HMQC sequence in order to measure long-range correlations: A so-called low pass filter eliminates signals of directly bound protons. It works because of the difference of more than one order of magnitude between 1JCH and
2,3
JCH . When the magnetization of
directly bound protons is in antiphase (after 1/[2 1JCH ]s), the small long-range couplings are still mostly in phase. A 90 pulse at this time converts the magnetization due to directly bound protons into DQC and ZQC but does not affect the signals of remote protons. A second, much longer delay brings the long range coupling into antiphase. From thereon the sequence is identical to HMQC. Because considerable signal is already lost due to relaxation during the long delay 1/[2
2,3
JCH ], no attempt at
13
refocusing is made (that would require a second delay of equal length), and no
C-broadband
decoupling is applied during t2. The line shapes are mixed absorptive/dispersive because the homonuclear 1H- 1H-couplings are of comparable size to the heteronuclear long range couplings. Therefore, the spectra have to be processed in magnitude or power mode.
5.6
Example for proton detectied 1H-13C shift correlation: HSQC

Coherence transfer pathway selection by gradients Menthol in acetone-d6 [Menthol] = 0.25 M Exp. time ca. 50 min.
ppm
20
30
40
50
60
70
3.5
3.0
2.5
2.0
1.5
1.0
ppm
5.7
Example for HMBC Coherence transfer pathway selection by gradients Menthol in acetone-d6 [Menthol] = 0.25 M Experiment time ca. 30 Min. (digital resolution in t1 reduced compared with HSQC)
ppm
20
30
40
50
60
70
3.5
3.0
2.5
2.0
1.5
1.0
ppm
6.1
6. Relaxation, dipole-dipole coupling and NOE

6.1 Longitudinal and transverse relaxation The longitudinal relaxation time T1 is the characteristic time constant describing the return of magnetization to Boltzmann equilibrium. This process requires energy exchange with the surrounding medium (in liquids, actually mostly other spins in the same molecule). For historical reasons (solidstate physics) this process is also called spin-lattice relaxation.
z Mz0
z t y M z(t) y x x t M 0
z
90 -x
y
Mz=0
Mz (t) Mz 0 = (Mz (t=0) M z 0 ) exp (t /T1) Mz = z-Magnetization at time t Mz 0 = z-Magnetization at Boltzmann equilibrium The longitudinal relaxation time is experimentally determined using the inversion-recovery pulse sequence:
z Mz
0
B relaxes fast z
negative signal z
positive signal Aquisition y
180
y x
0 M z
y x A relaxes slowly y
90 -x
x A B
A+B
(B) (A) 0 0 + Intensity
Crude estimate: Zero signal at = T1 ln 2 T1 = ca. 1.443 0
More precise results are obtained by fitting an exponential curve to the experimental points
6.2
Transverse relaxation The transverse relaxation time T2 describes the dephasing process of transverse magnetization. In an ideally homogeneous magnetic field, 1/T2 corresponds to the line-width of the signal. Relaxation mechanisms In order to be able to exchange energy with the "surrounding medium", the spins must have magnetic interactions that are modulated in time with the Larmor frequency of the excited transitions. For 1H and
13
C, the modulation of the dipole-dipole coupling by molecular tumbling is the dominating
mechanism. Other mechanisms that may contribute to systems in solution are the modulation of scalar couplings by fast conformational changes, the anisotropy of chemical shifts (CSA, e.g. carbonyl-13C at 10 Tesla) and quadrupolar relaxation. If the system contains unpaired electrons (e.g. transition metals or radicals), the dipole-dipole coupling with the much larger spin magnetic moment of the electrons dominates relaxation. Dissolved oxygen with S=1 can also contribute quite substantially to relaxation.
6.2 Nuclear Overhauser effect in a two spin system without scalar coupling The nuclear Overhauser effect (NOE) is defined as the change in the intensity of an "observed" spin I upon saturation of another spin S. fI {S} = (I - I0) / I0 A 50% increase of the signal intensity of I upon saturation of S corresponds to an NOE of 0.5. If saturation of spin S causes the signal of I to vanish, the NOE is -1.0. The relaxation mechanism that causes the NOE is dipole-dipole relaxation, which will be discussed in section 6.3. A simplified analysis of the NOE phenomenon (according to Noggle and Schirmer):
N
W1S W2IS N
W1I
W0IS W1I W1S
Transition probabilities: W1I and W 1S : single quantum transitions. W2IS: double quantum transitions W0IS: zero quantum transitions W0IS and W2IS are cross relaxation probabilities
6.3
Let us assume that, at Boltzmann equilibrium, the populations of the four states are N, N = N , N. The population differences are approximately (linearized Boltzmann): 2 = N N = N N = N N = N N Upon saturation of the S-transition, the populations of states connected by a transition of S are equalized: N s = N s and Ns = N s . Immediately after the onset of saturation of S, the populations will be:
Ns = N +
W1S W2IS Ns
W1I W0IS
=N
Ns
=N
W1I
W1S
N s = N
While the populations themselves change, the population differences for spin I, which are proportional to signal intensity, are still equal to those at equilibrium. Let us now examine the effect of relaxation along the four transition probabilities W 1S, W1I , W 0IS and W2IS . For each transition, the populations will change in the direction leading to re-establishment of the Boltzmann equilibrium. W1S has no effect because ongoing saturation by irradiation of S enforces equal populations for states connected by W 1S. Since the population differences Ns a N s and Ns Ns are still those of equilibrium, W1I has no effect on the signal intensity either. In contrast, the population difference across W0IS is now 2 (zero at equilibrium). Relaxation along W0IS acts towards reestablishment of the equilibrium populations by increasing NS let's say by 2 o and by decreasing NS by 2 o The population difference across W2IS is now N N 2, smaller than at equilibrium. Relaxation along W2IS acts towards reestablishment of the equilibrium population difference by increasing N S by 2 2 and by decreasing N S by 2 2 Because of the ongoing saturation, the newly acquired population differences due to W0IS and W 2IS are
6.4
immediately redistributed to give equal populations across the saturated transition. This gives the following populations:
Ns = N + +
0
1S
W W
2IS
1I
Ns
=N
+
0
W W1I
0IS
N s = N + +
0
1S
Ns = N +
0 2
The population difference across the transitions of spin I has changed by +22 20. Because of this deviation from the Boltzmann equilibrium, the relaxation process W1I will tend to change it back. Of course, all processes that have been discussed above in a stepwise manner are actually active simultaneously. Qualitatively, one comes to the following conclusions: It is the cross relaxation (W0IS and W 2IS) that generates the NOE. The intensity of signal I is increased by double quantum relaxation W2IS and decreased by zero quantum relaxation W0IS. Single quantum relaxation W 1I attenuates the NOE The formal mathematical treatment of the steady state NOE (Solomon equations) of a two-spin system leads to the following result for the steady state NOE on spin I upon saturation of spin S:
( W2 IS W0IS ) = S IS f I {S} = S W + 2W + W I ( 2IS I IS 1IS 0IS )

The term W2IS W0IS (= IS ) describes the contribution of cross relaxation to the relaxation rate of spin I. The denominator W2IS + 2 W1IS + W0IS (= IS ) corresponds to the total relaxation rate of spin I due to spin S.
6.5
7.3 The mechanism of dipole-dipole relaxation The transition probabilities W1IS, W 0IS and W2IS depend on magnetic interactions that fluctuate with the frequency of the corresponding transition. In solution, the modulation of the dipole-dipole coupling by molecular tumbling dominates relaxation; it generates a continuum of frequencies.
HI rIS B0
HS
The dipole-dipole coupling is dependent on the magnetic moments (s, I ) of the two coupled spins, on the distance between them, and on the orientation of the vector connecting the two spins relative to the magnetic field: The dipole-dipole coupling is proportional to
( 3cos 2 1) .
rIS
3
Because characteristic times for molecular tumbling are in the sub-nanosecond range, whereas dipolar couplings are in the 1-100kHz range, only the time average (<3 cos2 > = 1) is observed and dipolar couplings do not manifest themselves in liquids spectra.
1 cos2
1/ 3
6.6
The spectral density function describes the distribution of frequencies in the continuum of molecular tumbling processes:
Spectral density J() The correlation time c indicates the average time a molecule needs to rotate by 1rad (114 ) in any direction.
1/ c
log
Correlation time The correlation time is actually a tensor for all non-spherically symmetric molecules Example:
y x H H H z
The molecule will rotate much faster around the z-axis than around the y-axis ("paddle" effect). Hence, cz is distinctly shorter than cy and the relaxation times of para- and ortho-protons are quite different. In practice, the available data usually do not allow doing a full tensor calculation and one has to work with the approximation of a single scalar correlation time. However, in the case of larger molecules that have mobile side chains, it is necessary to take the shorter correlation times of the flexible parts into account. Example: Methyl groups in large molecules such as proteins or nucleic acids.
H r'IS rIS
The methyl group rotates around the -bond much faster than the molecule as a whole re-orients itself in solution.
H H
6.7
For a rigid, spherical molecule, the correlation time can be estimated using the following equation: c = 4a 3 3k T
= viscosity of the solution (solvent) a = molecular radius
c [s] = 1.011012 [cP]a3 [ A3 ] for 25C

Crude estimate: C = 10 12 s . molecular weight (for CDCl3 at 25C)
The best way to determine correlation times experimentally is by measuring the relaxation time T1 of
13
C-nuclei (in well-degassed solution) and by using the following equation: c = 2.8110 11 NT1
{ C}
13
N = number of hydrogens directly bound to the carbon
Transition probabilities W1I, W2IS and W0IS as a function of and c The corresponding expressions are the following::
W0IS W2IS
2 1 I S c =c 3 2 r 10 IS 1+ ( I S ) c2 2 3 I S c =c 3 2 r 5 IS 1 + ( I + S ) 2 c
2 3 I S c W1S = c 3 r 1 + 2 2 20 IS S c 2 3 I S c W1I = c 3 r 1 + 2 2 20 IS I c
For the homonuclear case (1H- 1H-NOE), I and S are almost the same and I = S. By substitution of the expressions for the transition probabilities into the general equation for the two-spin NOE one obtains the following relationship for the homonuclear NOE:
f I {S}max =
5 + 2 2 4 4 c4 c 10 + 23 2 c2 + 4 4 4 c
6.8
Considering this equation we can draw the following conclusions:

1. 2. For c << 1 (fast molecular tumbling), the NOE assumes its maximal value of 0.5. For c >> 1 (very slow molecular tumbling), the 4 th order term dominates and the NOE assumes the negative limit of 1.0 (e.g. extinction of the signal of spin I) 3. 4. For c = 1.12, the NOE vanishes. If dipole-dipole relaxation is the only relaxation mechanism, the two-spin NOE does not depend on the distance of the two nuclei at all. 5. The zero crossing depends on both, and C. Therefore, the region in which zero or negative NOEs will be observed shifts towards smaller molecules with increasing magnetic field of the spectrometer. The range of correlation times where c << 1 is called the "extreme narrowing range".
7.1
7. Homonuclear 1D-NOE-difference spectroscopy

7.1 Steady state NOE in homonuclear multi-spin systems
In a multi-spin system, the NOE observed on the signal of spin I when spin S is saturated is given by the following equation:
6 6 rIS f X {S}rIX 6 6 rIS + rIX X X
f I {S} = fmax
This equation holds under the assumption that the relaxation mechanism is exclusively dipolar and that a single correlation time describes the molecular motion. f max is the maximal two-spin NOE given by the equation on page 6.7, e.g. 0.5 for the extreme narrowing range. Conclusions from this equation: 1. The NOE fI {S} in multi-spin systems consists of two parts: a direct and an indirect contribution. The direct part (first term in the nominator) represents the contribution of cross-relaxation between spins I and S. The indirect part (second term in the nominator) is the sum of all contributions from any other protons X (XI,S) that change their populations because they exhibit a NOE f X{S}. The negative sign of the indirect term indicates that this contribution opposes that of the direct one. Because any NOE fI {S} depends on the NOEs fX{S} of all other protons, the equation can not be solved in analytical form but must be evaluated by iteration. 2. The denominator of the equation represents the total dipolar relaxation rate of spin I. As for the two-spin system, the NOE can be interpreted as the ratio between the cross relaxation rate and the total relaxation rate. 3. Without other relaxation pathways through protons X, the NOE fI {S} would be independent of the distance rIS . In reality, experimental steady state NOEs are usually much smaller than predicted by the above equation. This is due to the contribution of relaxation mechanisms other than dipole-dipole such as: - Electron-nucleus dipole-dipole relaxation from traces of transition metals and dissolved oxygen. - Scalar relaxation due to dynamically modulated coupling constants - Spin-rotation relaxation - Chemical shift anisotropy relaxation - Quadrupolar relaxation or modulation of couplings to quadrupolar nuclei. - Intermolecular dipolar relaxation.
7.2
Since all these other relaxation pathways only contribute to the total relaxation rate of spin I, but not to the cross relaxation rate, they are commonly grouped as "leakage rate" L I and added to the denominator:
f { S} r 6 f { S} = fmax 6 r 6 + r + L
r 6 IS
IS X IX X I IX I X
The attenuation of the NOE by leakage is particularly large for NOEs between distant protons (small rIS -6). In order to detect such weak NOEs one has to degas the solution and sometimes needs to extract the solution with EDTA in order to remove traces of transition metals. Indirect NOEs: the three-spin system Because the indirect and direct contributions have opposite effects, there are some geometrical constellations that exhibit a net NOE of zero, although all protons are quite close to each other and c is in the extreme narrowing range.
S 60 1.5
1.5 60
X Predicted without leakage: fI {S} = 0.21 Contributions: + 0.25 direct and 0.04 indirect via X
1.5 60 I 1.5 78
X Predicted without leakage: f I {S} = 0 1.5 Contributions: + 0.11 direct and 0.11 indirect via X
1.5 S
X 180
1.5
Predicted without leakage: fI {S} = 0.13 Contributions: + 0.01 direct and 0.14 indirect via X
7.3
Important conclusion: The absence of a steady state NOE can never be taken as a definite proof for structure! Individual NOEs may be weakly negative, although the correlation time is in the extreme narrowing range. In such cases, the negative NOE may be an indication of a quasi-linear arrangement between the spins I, X and S. Attention: saturation transfer (see below) may also give negative signals in the extreme narrowing range.
7.2 1D-NOE-difference spectroscopy The basic NOE experiment measures the change in the integral of signal I when spin S is saturated by selective irradiation. In contrast to decoupling, which acts immediately, the build up and the decay of the NOE take times in the order of T1. Therefore, FT-NMR allows to generate the steady state NOE during a pre-irradiation time and then switch the irradiation off for the acquisition. This has the advantage that neither decoupling of scalar couplings nor the so-called Bloch-Siegert shifts are active during the acquisition. The best way to visualize the NOEs and to cancel out any other instabilities, is to carry out a difference experiment: the FID acquired with irradiation in an empty region of the spectrum is subtracted from the FID acquired with the same irradiation power and duration but on-resonance (at the frequency of the signal of spin S). Such difference spectra allow to observe much weaker NOEs (ca. 0.05%) than by comparative integration (ca.2%). Pulse sequence:
non-selective pulse
selective CW irradiation ca. 2-5s
acquisition
Practical aspects: Typically, one acquires 8 or 16 scans on-resonance, and then the same number of scans off-resonance and stores the two FIDs independently. This alternating block is repeated in a loop as long as needed to generate good signal to noise for the difference signals. Since we want to observe signals as weak as 1% of the normal 1D-spectrum, it is necessary to measure for a much longer time than for 1D spectra! If several NOEs have to be measured for the same sample, up to five different on-resonance irradiations can be combined with a single off-resonance reference in one loop, which reduces the measuring time considerably.
7.4
Selectivity: in crowded spectra it is sometimes difficult to saturate one signal without affecting those nearby. Because inadvertently co-irradiated signals can falsify the interpretation, it is preferable to reduce irradiation power and sacrifice part of the saturation in favor of selectivity. If a signal is only saturated to 80%, the corresponding NOEs will simply be scaled down proportionally. Relatively broad multiplets, if irradiated at the center frequency, need much more power for uniform saturation than singlets. In this case it is better to irradiate each sub-line separately with low power and to add the result (see SPT below) than to try to saturate the multiplet with a single frequency setting. Another method is to irradiate all sub-lines of a multiplet in time sharing by cycling the frequency among the sub-lines during the irradiation time (frequency hopping).
selective saturation with a single frequency is impossible
selective saturation of individual sub-lines with less power
Like all difference methods (2D with phase cycles etc.), 1D-difference NOE experiments are very demanding in terms of the stability of the spectrometer and the surroundings. Processing: It is preferable to subtract the FIDs and to transform the resulting difference FID (which transforms to the difference spectrum). Subtracting transformed spectra is inaccurate because of tiny phase- and baseline errors. In order to improve cancellation of sharp signals, a modest line broadening of 1-2 Hz is usually applied to the FID before transformation. In the difference spectrum, the irradiated signal is a large negative signal (-100%); NOEs appear as weak positive signals (for molecules in the extreme narrowing range).
Problems and Artifacts: 1. Saturation transfer If the saturated proton S slowly ( > k > 1/T1) exchanges with another proton S', saturation will be transferred to the signal of S' during the irradiation time. This phenomenon, called saturation transfer, can be recognized from large negative signals not only at the place of irradiation, but at the chemical shift of other protons as well. This situation is equivalent to saturating all exchanging signals together and the NOEs that appear can be caused by any of the exchanging magnetic sites. In particular, coirradiation of the tail of a broad signal from an exchanging proton can easily lead to misinterpretation:
7.5
proton that is close in space to OH
irradiated signal
-OH H2 O
chemical exchange false apparent NOE saturation transfer
co-irradiated
actual NOE
Note: Saturation transfer (or its 2D variant EXSY) can be a very useful tool to study exchange processes in cases where exchange is too slow to give coalescence. 2. Selective population transfer (SPT) If the individual sub-lines of a multiplet are not uniformly saturated, the 1D NOE difference spectrum shows strong antiphase signals at the chemical shift of the scalar coupling partner(s) of the irradiated proton.
SaturationofA1 Ns = Ns
Ns = N + X1
A1
Ns
=N
X2 A2
Saturation of A1 increases the intensity of X2 and decreases the intensity of X1
7.6
Difference spectra: a) A 1 saturated X2 b) A2 saturated X1
A1 a)+ b)
X1
A2 a) - b) A2
X2
X X
1
No SPT X1 A1 A2 A1 only SPT antiphase
Saturation of each sub-line in a separate experiment, followed by addition of the resulting FIDs, makes the SPT effect disappear and only the NOE remains. On the other hand, subtraction of the two FIDs gives a pure SPT spectrum. The latter can be used to locate coupling partners in strongly overlapping spectra (as an alternative to homo-decoupling). 3. Spin diffusion For very large molecules (C >> 1.12), all NOEs are negative. In this regime, all NOEs build up very rapidly and indirect NOEs can no longer be distinguished from direct ones (saturation of a single signal of a large protein for 5s will lead to disappearance of the whole spectrum; NOE = 1). This rapid propagation of population differences through a molecule is called spin diffusion. Therefore, the 1D-NOE-difference method is not suitable for large molecules and kinetic variants of NOE experiments have to be used instead.
7.7
Rules for planning and interpreting 1D-NOE difference experiments 1. 2. 3. All proton signals have to be assigned correctly, before a NOE experiment makes sense. Selectivity is more important than full saturation Steady state NOEs are not symmetric (fI {S} fS{I}): it is preferable to irradiate groups with several magnetically equivalent and geometrically close protons (such as a methyl- or t-butyl group) and to observe NOEs on protons that are geometrically isolated. 4. Measure as many NOEs as possible and check for consistency. The absence of an NOE is no proof for large distance. 5. Check in the difference spectrum whether other signals have been co-irradiated or exhibit saturation transfer (strong negative peaks). 6. Identify the SPT effects by checking signals of coupling partners. If the integral of the signal is clearly positive, an NOE may be overlaying an SPT effect (pure SPT signals have integral zero). 7. The limit of confidence must be estimated from the residual signals of protons that can not exhibit an NOE. NOEs observed on t-butyl- or methyl groups must be strong to be distinguishable from imperfect cancellation; NOEs on broader multiplets are easier to recognize. 8. Always use a molecular model when analyzing NOEs. For molecules that may assume several conformations, all must be considered in the interpretation (see chapter 9: NOE and dynamic processes) 9. NOEs may be integrated and quantitatively specified relative to the integral of the irradiated peak (-100%). However, reproducibility of absolute NOE intensities is poor because of the leakage rate, which may vary from one sample to the other even for identical substances. In order to prevent over-interpretation, it is prudent to classify the NOEs into a few categories such as s (strong), m (medium) and w (weak).
8.1
8. Kinetic NOE-spectroscopy
The NOE effect needs time to build up when the population of a nearby proton is changed either by saturation or by inverting its populations with a 180 pulse. The kinetic equation for the build up of the NOE upon saturation of S is: dIz = RI (Iz I0 ) IS (S z S 0 ) z z dt
IX (Xz X0 ) z
X
Because Iz = Iz 0 and Xz = Xz 0; Sz Sz 0 = Sz 0, the build-up rate at t=0 is: dIz [ t = 0] = ISS0 z dt In other words, the initial build up rate of the NOE is only dependent on the direct cross-relaxation term IS . Both, total relaxation of I and indirect NOEs (spin diffusion), have no effect at t=0. If the build up rate of the NOE is measured instead of the steady state NOE, distance information can be derived from NOE experiments even for large molecules in the regime c 1.12 . Because IS = c / r IS 6 it is possible, at least theoretically, to derive unknown distances arks from the cross relaxation rates and the known distance of a calibration pair (e.g. ortho-protons at aromatic rings):
6 IS rIS = 6 KS rKS
TRNOE (transient 1D-NOE) Instead of saturating S, the signal of S is inverted by a selective 180 pulse. Therefore, the magnetization of S at t=0 is Sz (t=0) = Szo which gives twice the build up rate obtained with saturation of S. In order to measure the build up curve of the NOE, the experiment has to be performed for a series of incremented delays .
TRNOE 180 selective wait for full relaxation (> 5 T ) 1 90 non selective
Aqu
The TRNOE is symmetrical ( I {S} = S{I}). In contrast to the steady state NOE, the transient NOE builds itself up, goes through a maximum and decays to zero with increasing delay time .

0
8.2
Iz I z
dIz /dt ( = 0)
direct NOE
indirect NOE
Indirect NOEs can be identified because, like an intermediate in reaction kinetics, they build up only after the direct NOEs and give a sigmoid build up curve. Problems: Because the initial build-up rate corresponds to the tangent to the build-up curve at t=0, one has to measure several data points at short . At these early times, the NOEs are still very weak and very long accumulations are necessary to get good S/N. The experiment time is further lengthened because full relaxation has to be allowed between scans. Since the result of 1D-TROE is identical to that of NOESY, the latter has largely replaced 1D kinetic NOE spectroscopy nowadays.
8.1 NOESY
The best NOE experiment for large molecules in the c 1.12 regime is NOESY. As a 2D-method it has the additional advantage of giving all NOEs in the molecule in one experiment. Pulse sequence: 90 90 90
relaxation
t1
tm
Acquisition
After excitation and labeling the signal with chemical shifts during t1, the magnetization is reconverted to z-magnetization. The cross relaxation processes act during the mixing time on the differently populated states, which are then reconverted to transverse SQC for acquisition. The mixing time t m plays the same role as the delay in TROE. In the NOESY spectrum, all cross peaks and the diagonal peaks are purely absorptive. For c 1.12, the cross peaks have the same phase as the diagonal peaks. This makes it impossible to
8.3
distinguish exchange ("saturation transfer") cross peaks from NOE cross peaks. For c 1.12 (positive NOE, "extreme narrowing range"), NOE cross peaks and diagonal peaks have opposite phase, while exchange cross peaks have the same phase as the diagonal. However, theoretical cross peak volumes are distinctly lower in the extreme narrowing range than under the c 1.12 regime. Therefore, for small molecules, 1D-NOE-difference spectroscopy is usually the better choice than NOESY provided that selective irradiation is possible. For large molecules, spin diffusion ("indirect NOEs") can only be recognized if several NOESY spectra with different mixing times are measured. The plot of cross peak volume vs. mixing time then allows to construct the build-up curves which reveal indirect NOEs through their sigmoid shape (see diagram for TROE above). Artifacts: Modulation by scalar coupling during the mixing time tm can lead to COSY type cross peaks, which can be recognized by their antiphase pattern. NOE build-up is much slower than the modulation through scalar coupling. Therefore, COSY type artifacts can be eliminated by a small random variation of the mixing time by 5-10%, which smears the COSY modulation by destructive interference. For short mixing times (<80ms) and for quantitative work, this random variation is not recommended.
8.2 NOE in the rotating frame: ROESY

Many classes of important natural compounds such as Oligosaccharides, Oligopeptides and Oligonucleotides have correlation times which lead to very small or even vanishing NOEs on high-field spectrometers (c near 1.12). In such cases, one has to use transverse NOE-spectroscopy (ROE = rotating frame Overhauser effect). The 1D variant is called CAMELSPIN, the corresponding 2Dmethod is ROESY. In contrast to longitudinal (exchange of z-Magnetization) NOEs, transverse NOEs (exchange of spin locked xy-magnetization) are always positive, regardless of the correlation time. The pulse sequence of ROESY is identical to that of TOCSY. However, in order to avoid TOCS type transfer, a much weaker (2-5 kHz) CW spin lock is used for ROESY, whereas for TOCSY, a strong (10-12kHz), composite pulse-decoupling field is best.
ROESY 90 tm t1 CW-Spinlock t2
8.4
ROE +0.6
+0.5
+0.4
+0.3 0.1 1 10
As can be seen from the diagram above, the phase of ROE cross is opposite to that of the diagonal. Exchange cross peaks and indirect ROE cross peaks have the same phase as the diagonal. Therefore, chemical exchange and spin diffusion can be recognized much more easily in ROESYthan in NOESY-spectra. Because the theoretical ROESY cross peak volumes for small molecules are much smaller than for large molecules, 1D-NOE-difference spectroscopy is preferable for small molecules (c << 1.12) and ROESY is only the method of choice near (c = 1.12). Artifacts: ROESY is probably the most artifact-ridden one of all 2D methods. The volume of a ROESY cross peak is not only a function of the ROE but also of the frequency offset of the two signals. As with NOESY, scalar coupling leads to COSY type antiphase cross peaks, in particular at short mixing times. Since the pulse sequence is identical to TOCSY, coupled protons may show TOCSY cross peaks (in phase with diagonal). Because TOCSY and ROESY cross peaks have opposite phase, TOCSY effects often cancel ROE cross peaks partially or entirely. TOCSY-ROE combinations are particularly tricky, because they have the same phase as true ROEs. A TOCSY transfer between strongly coupled protons A and B, in combination with a ROE between B and C gives a false ROESY peak between A and C: Apparent ROE AC
ROE
H TOCSY H ROE H H
J
H
JAB > 0
False ROE
9.1
9. On the influence of dynamic processes on the NOE

9.1 Overall molecular tumbling, internal rotation (conformational changes) and chemical exchange Exchange The interpretation of NOE data is straightforward only for the rather rare cases of conformationally rigid molecules. If several conformations are significantly populated, one has to consider the influence of dynamic conformational equilibria on the NOE results. Conformational dynamics due to rotation around single bonds are usually so fast that the time-averaged NMR spectrum is observed (rapid exchange with respect to chemical shift differences and coupling constants). In this case, the chemical shifts and coupling constants are population-weighted averages and NOE experiments on the individual conformers are impossible. At room temperature, rotation around partial double bonds such as in amides is often slow enough to give separate sub-spectra for the individual rotamers. Technically, it is then possible to irradiate signals and to measure the NOE effect for individual rotamers. The rates of chemical exchange (when bonds to protons are broken and reformed by the dynamic process) such as proton exchange between -OH and NH groups vary over many orders of magnitude. They depend not only on the pKa of the exchanging groups but also on the nature of the solvent, ionic strength and pH. With regard to the NOE it is only of technical interest, whether the dynamic rate is high enough to lead to rapid exchange spectra or not. Below coalescence, it is possible to irradiate and observe signals belonging to individual components whereas above coalescence, this is obviously impossible. The factor determining whether and how NOE effects are averaged is the ratio between the rate of the dynamic process and the inverse of the two characteristic time constants T1 and C. If k << 1/T 1, the conformers / rotamers behave like a mixture of substances and correct NOEs for each component are obtained with 1D-NOE-difference, NOESY, and ROESY. The absence of saturation transfer in 1D-NOE-difference spectra and of exchange peaks in NOESY and ROESY spectra is a clear indicator for this time regime.
9.2
coalescence only the averaged spectrum is observed k >> k [s -1 ] k >> 1 /

c
spectra of individual forms observed k <<
1 / T << k << 1/
1
k << 1 / T
Average of dipole-dipole coupling < r-3 >

2
Average of cross relaxation rates < >= < c > < r -6 >
non averaged NOEs: Behaves like a mixture of substances
IS
For 1/c >> k >> 1/T 1 the resulting averaged NOE must be calculated using time averages for the relaxation rates: fI {S } = IS RI IS = RI =
fx {S}
x k
IX RI
k x kIS
xkRIk
k
xk molar fraction of component k IS k , RI k (cross) relaxation rates for component k Below coalescence, such cases can easily be identified from the appearance of saturation transfer (1D) or exchange peaks (2D). Typical cases are rotations around partial double bonds, in particular, of amides. Above coalescence, this time regime of averaging is much more difficult to recognize. Most often, it is the observation of NOEs not compatible with a single conformer or with observed scalar coupling constants that forces us to conclude that several conformers must be significantly populated. Typical - and very frequent - cases are conformational changes such as ring inversions, rotation of sterically hindered phenyl rings or t-butyl groups, host-guest association equilibria and substrate binding to enzymes. In this time regime, the dynamic process changes the NOE only, if the inter-nuclear distances are changing during the interconversion of the different forms. Whereas chemical shifts and coupling constants are linearly averaged according to molar fractions, the NOE average over (1/r IS )6 gives very non-linear behavior with regard to distance. A very minor component with a short inter-nuclear distance will contribute significantly to the observed NOE even if this distance is long in the major conformer. Example:
9.3
Hb O R Ha
Ha
I
Hc
O
II
Hc
As long as R is relatively small, rotation around the partial double bond is slower than overall molecular tumbling but fast enough to give coalescence. In order to calculate the NOEs, one has to weight the relaxation rates <ab>, <ac > and <Ra>, <Rb>, <Rc > with the molar fractions of I and II. Although the s-cis-conformer is the minor one, it has a shorter distance Ha-H b than II and will lead to an apparent contradiction between the NOEs H aHb and HaHc if only the major conformer is considered in the analysis. In principle, the equations shown above would allow to determine the molar fractions of individual conformers from a large enough set of NOEs (and, possibly, coupling constants). The problem is, however, that the precise geometry and therefore the inter-nuclear distances of the individual conformers are generally not known. This makes the analysis of dynamically equilibrated multiconformation situations by NMR extremely difficult; it is one of the weak points of NMR. If k >> 1/C (c = correlation time for the molecule as a whole), the time average must be calculated over the dipole-dipole couplings rather than over relaxation rates. IS = c c r3
2
In this case, the NOE is influenced even if no inter-nuclear distances are changed at all by the dynamic process. Typical cases are flexible side-chains or freely rotating methyl groups of large molecules. Empirically, it has been shown that neglecting this process for methyl groups leads only to a small error if the individual distances to the three protons of a methyl group are replaced by the distance to their center of gravity.
0 0 H=FF 0
9.4
9.2
How to proceed with molecules with oc 1
1D-NOE-difference spectrum: look for a NOE that must be there for constitutional reasons (e.g. ortho-protons on aromatics)
NOE positive and large
NOE negative and large
NOE very small or zero
higher temperature lower viscosity solvent lower field
lower temperature higher viscosity solvent higher field
1D-NOE difference
ROESY
NOESY
10.1
10. Combined application of several methods

10.1 Strategies for solving typical organic structure problems General considerations: NMR parameters, in particular chemical shifts and exchange rates, can be strongly dependent on temperature, solvent, pH, ionic strength and concentration. Therefore, assignments can only be carried over from one spectrum to the other if all experiments have been taken under the same conditions. It saves much time to optimize the conditions carefully on the 1D-spectra, before starting the series of 2D and heteronuclear spectra, because changing solvent, temperature or concentration later, might make it necessary to repeat all experiments. In addition, 1D-spectra allow to recognize the presence of slow dynamic processes such as the equilibrium between several rotamers below or near coalescence or line broadening due to slow chemical exchange. Lines broadened by dynamic processes (13 C and 1H; short T2) can make the acquisition of DEPT and heteronuclear shift correlation spectra impossible. Solvents: The optimal selection of solvent is very important. In non-polar solvents such as CDCl3, signals of exchangeable protons are often broad and their appearance depends strongly on residual water and traces of acids or bases. CDCl3 and (more slowly) CD2Cl2 develop DCl upon standing. They should be filtered through neutral alumina and stored in the dark before use. In order to have sharp -OH signals showing couplings to the vicinal protons, dry [D6]-DMSO is the solvent of choice. Substances, which are only soluble in protic, polar solvents, will loose all exchangeable protons due to exchange with deuterons of the solvent. If the signals of these protons are important for structure elucidation, one has to measure in the non-deuterated solvent (H2O/D2O 9:1 or CD3OH etc.) and suppress the solvent signal. For slowly exchanging -NH protons (k << 1 s1 ), solvent suppression can be done with presaturation, for faster exchange, zero-excitation methods such as WATERGATE or excitation sculpting have to be used. In protic solvents, -OH protons are usually exchanging fast enough to give coalescence and can not be detected even with zero-excitation. Be aware of the possibility that, within the long time an NMR-sample stays dissolved in a protic deuterated solvent, exchange of protons by deuterons can also occur for acidic CHn groups with pKa >16. Carbons carrying only deuterium atoms have extremely long relaxation times and are split into several lines. If such an exchange has taken place, not only the signal of the exchanged proton but also the corresponding carbon signal will usually be missing in the spectrum. S/N and sample concentration needed for general structure elucidation work: With a modern spectrometer (e.g. 500MHz 1H, 1H{X}-inverse probehead, X {1H}-probehead, gradient unit), all necessary spectra can be taken within reasonable time if 10 mol of the pure compound are available. With a standard 5mm tube, filled with 0.6ml solvent for easy shimming, this gives a 16mM
10.2
solution. The practical lower limit for a full structure elucidation using standard 5mm probeheads and tubes and a 500 MHz 1H spectrometer is at ca. 2 mol. This requires one weekend for the 13 Cspectrum and ca 24h for the HMBC spectrum. Means to increase S/N without going to very high field (700, 800, 900 MHz) Shigemi-tubes With special susceptibility-matched NMR tubes (Shigemi-tubes, $100/tube, available for H2O, Methanol, DMSO and CDCl3), the sample volume can be reduced to ca. 250 l, gaining a factor of ca. 2 in the signal to noise. Cryoprobes: Recently, probeheads have been developed in which the RF coils and electronics parts are cooled to near liquid helium temperature while the sample stays at normal temperature. This eliminates most of the thermal noise in the receiver channel and improves S/N by a factor of ca. 3 to 4. However, apart from the considerable costs of this equipment, the operation of cryoprobes does not allow probehead changes within reasonable time and more or less requires dedicating one spectrometer to the operation of one cryoprobe. While already in regular use for Bio-NMR, such probeheads are far from being standard equipment in regular organic chemistry NMR labs. Microprobes Special probeheads using very small sample tube diameters (ca. 1mm) have been developed. They have an active volume of ca 5 l and give a dramatic improvement of S/N if the total amount of sample and not solubility is the limiting factor. This type of probehead is becoming popular in laboratories specialized in the isolation and structure elucidation of secondary metabolites from nature. Ab initio structure elucidation of an unknown compound: First Task: Constitution Ab inito elucidation of the constitution means to establish the molecular formula and the connectivity between all atoms. Minimally, one needs to know the molecular weight (M+ ) or better, the molecular formula (obtainable through high resolution MS), before starting the NMR work. The best-suited NMRmethods are those which allow to determine connectivity via direct and long-range scalar coupling. The spin systems of NMR active nuclei such as 1H,13 C, 31 P ,( 15 N, 19 F) can be identified and connected via long range couplings. Scalar coupling to quadrupolar nuclei such as 14 N or
11
B is usually not
resolved due to fast quadrupolar relaxation. The presence and location of NMR inactive or fast relaxing quadrupolar nuclei such as O, S, Cl, Br, I has to be inferred from MS data and chemical shifts of attached carbons and protons.
10.3
The following table gives typical experiment times for good quality spectra with 10mol of substance: Method Time [h]
1
Information
Remarks
H 1D C-BB{1H} C DEPT90/135
0.5 6 3 1.5 1.5 3
H, JHH -values for isolated signals Number and C of carbons CH, CH2, CH3 Scalar couplings, 1H-spin systems H-C shift correlation trough 1JHC H-C shift correlation trough 2,3JHC
Integration, symmetry Incl. quaternary C, symmetry? # of 1H not bound to heteroatoms Large/small active JHH Non equivalent CH2 Incl. correl. across heteroatoms
13
13
DQF-COSY.grad HSQC.grad HMBC.grad
Depending on the type of molecule, the following additional methods may be useful: Method Time [h]
1
Information
Remarks
H 1D after D 2O
0.5
Identification of exchangeable 1H (-OH, -NH, -SH etc.)
non protic solvents only
exchange
1
H 1D with
Identify exchangeable 1H in protic solvents. NOEs and coupling information on exchangeable NH
use H2O/D2O 9:1 or CD3OH etc. Solvent suppression: presaturation or zero excitation depending on exchange rate
solvent suppression
31
P 1D with and
0.5
Number and type of
31
P-atoms
for sharp 31 P-signals: JPH, look at 13 C for JPC.
without 1H dec. H-P-COSY TOCSY 4 6 H-P correlation via 2,3JHP whole 1H-spin systems
e.g. for oligonucleotides e.g. oligopeptides, -nucleotides, -saccharides
H-N-HSQC.grad
H- 15 N-shift correlation for nitrogens
e.g. amide NH, imino-NH, pyrrole NH.
bound to slowly exchanging 1H
10.4
Interpretation Use the chemical shifts from the 1H and 13 C 1D-spectra to identify possible functional groups. At this early stage, chemical shifts should only be used qualitatively by subdividing the spectra into course ranges of chemical shifts:
1
H: aliphatic protons < 2 ppm; protons vicinal to carbonyl or double bond 2-3 ppm; protons geminal to
O: 3-4 ppm; Protons at double bonds 4-6 ppm; protons geminal to two heteroatoms 5-6 ppm; protons at aromatic rings 6.5-8 ppm etc.
13
C: aliphatic sp3 10-40 ppm; bound to N sp3: 30-40 ppm; bound to O sp3: 50-60 ppm; bound to two
heteroatoms 90-110 ppm; double bond and aromatic sp2: 110-140 ppm; sp 2 bound to heteroatom: >130 ppm; carbonyl 160-220 ppm etc. Identify exchangeable protons, e.g. by D2O exchange. Using the information from the 1D-spectra and DEPT, establish a first balance of the number of protons bound to carbon, on the number of C, CH, CH2, CH3 and whether these carbons carry heteroatoms. Compare all this information with the molecular formula and identify inconsistencies. Number the carbon signals according to chemical shifts. List all carbons with attached protons and heteroatoms. Use the HSQC or HMQC to carry carbon numbering over to protons. For non-equivalent CH2-groups use primes (e.g. 8' for the proton at higher chemical shift and 8" for the one at lower chemical shift). From DQF-COSY (TOCSY) establish the coupling network for each isolated 1H-spin system: identify geminal cross peaks from the information in HSQC (non-equivalent CH2-groups). Qualitatively estimate the coupling constants from cross peaks. Be aware of the possibility that small couplings may be long range couplings. Collect a table of fragments: E.g.
H3 C
H3 C
CH
C C C H2 H2
X=O, ev. Cl
3 OH
1 (CO?) NH
Compare again with the molecular formula. Be aware of the possibility that a given heteroatom may occur in several fragments (ether, ester, amine, and amide linkage). Try to connect the fragments using the information from HMBC. This leads to one (in favorable cases) or several possible proposals for the constitution.
10.5
Second task: relative configuration at stereogenic centers. Geometrical information obtained from vicinal coupling constants (via Karplus relation) and NOEs always pertains to a given conformation. Therefore, relative configurations can only be determined by NMR via a conformational analysis and if the molecule exists predominantly as a single conformer. This is usually the case for polycyclic or six-membered ring partial structures but not for open chain segments. Five membered rings (and six membered rings containing two or more sp2-centers) are borderline cases that may be amenable to analysis if a full set of coupling and NOE data is available and proves to be consistent with a single predominant conformation. It is often possible to convert open chain molecules into cyclic systems such as lactones, lactams, ketals etc. with a little chemistry. This approach is much more secure and often faster than to try to deduce relative configurations at open chain stereocenters from NOE and coupling constants. The E/Z configuration at double bonds can usually be determined by NMR (HC=CH coupling constant in 1,2 disubstituted double bonds, NOEs in tri- and tetra-substituted double bonds). If rotation around partial double bonds is slow, NOEs often allow determining whether they are predominantly cis or trans. In any case, one has to keep in mind that the NMR data might by time averages over several populated conformers. Clear signs for such cases are: strongly temperature dependent chemical shifts and coupling constants. a set of coupling constants and NOEs incompatible with a single conformation
Because such inconsistencies can only be recognized from a set of redundant data, it is very important to try to measure as many coupling constants and NOEs as possible. Absolute configuration The absolute configuration can not be determined by NMR directly. Through formation of diastereoisomeric complexes (e.g. with chiral lanthanide shift reagents) or with covalent derivatives such as Mosher esters and comparison with the corresponding derivatives of the pure enantiomers it is possible to assign the absolute configuration or - more typically- to determine enantiomeric excess. Partial structural problems During synthetic work, it is rare that the constitution of the product molecule is completely unknown. Apart from unexpected rearrangements etc., the problem is normally reduced to verifying the expected structure and solving questions of regioselectivity or diastereoselectivity. In addition to the standard
1 13
H-,
C- and DEPT spectra, this may necessitate DQF-COSY and HSQC in order to allow secure
assignment of all proton and carbon signals for publication. The determination of the relative configuration at certain stereogenic centers may make it necessary to measure a series of 1D NOEs as well.
11.1
11. References
11.1 Textbooks
Theoretical treatment: R. R. Ernst, G. Bodenhausen, A. Wokaun, Principles of nuclear magnetic resonance in one and two dimensions, Clarendon Press, Oxford, 1987. A good introduction at a similar level than this course: T.D.W.Claridge, High Resolution NMR Techniques in Organic Chemistry, Pergamon, 1999. Simpler, with less 2D: J. K. M. Sanders, B. K. Hunter, Modern NMR Spectroscopy, Oxford University Press, Oxford, 2nd Edition, 1993. Attention: the 1st Edition is completely outdated. H. Friebolin, "Ein- und zweidimensionale NMR Spektroskopie", 3rd. Ed. Wiley VCH, 1999 H. Friebolin, "Basic one and two dimensional NMR spectroscopy", 3rd Ed. VCH 1998. Reviews: H. Kessler, M. Gehrke, Ch. Griesinger, "Two-Dimensional NMR-Spectroscopy: Background and Overview of the Experiments", Angewandte Chemie (Int. Ed. Engl.) 1988, 27, 490-536. Review of 2D spectroscopy including proton detected and gradient methods; practical tips: Two-Dimensional NMR Spectroscopy, Applications for Chemists and Biochemists, Ed. W. R. Croasmun, R. M. K. Carlson, VCH Publishers, Weinheim, 2nd Edition, 1994.
11.2 COSY
Homonuclear correlation through scalar coupling
W. P. Aue, E. Bartholdi, R. R. Ernst, J. Chem. Phys. 1976, 64, 2229-2246. A. Bax, R. Freeman, J. Magn. Reson. 1981, 44, 542-561. DQF.COSY U. Piantini, O. W. Srensen, R. R. Ernst, J. Am. Chem. Soc. 1982, 104 , 6800-6801. M. Rance, O. W. Srensen, G. Bodenhausen, G. Wagner, R. R. Ernst, K. Wthrich, Biochem. Biophys. Res. Commun. 1983, 117, 479-485. A. J. Shaka, R. Freeman, J. Magn. Reson. 1983, 51, 169-173. N. Mller, R. R. Ernst, K. Wthrich, J. Am. Chem. Soc. 1986, 108 , 6482-6492. E.COSY C. Griesinger, O. W. Srensen, R. R. Ernst, J. Magn. Reson. 1987, 75, 474-492.
11.2
C. Griesinger, R. R. Ernst, J. Magn. Reson. 1987, 75, 261. SOFT.COSY R. Brhschweiler, J. C. Madsen, C. Griesinger, O. W. Srensen, R. R. Ernst, J. Magn. Reson. 1987,
73, 380-385.
TOCSY L. Braunschweiler, R. R. Ernst, J. Magn. Reson. 1983, 53, 521-528. A. Bax, D. G. Davis, J. Magn. Reson. 1985, 65, 355-360. D. G. Davis, A. Bax, J. Am. Chem. Soc. 1985, 107 , 2820-2821. 1D-TOCSY H. Kessler, U. Anders, G. Gemmecker, S. Steuernagel, J. Magn. Reson. 1989, 85, 1-14. F. Inagaki, I. Shimada, D. Khoda, A. Suzuki, A. Bax, J. Magn. Reson. 1989, 81, 186-190. INADEQUATE A. Bax, R. Freeman, S. P. Kempsell, J. Am. Chem. Soc. 1980, 102, 4849. J. Buddrus, H. Bauer, Angew. Chem. 1987, 99, 642-659. R. Dunkel, C. L. Mayne, R. J. Pugmire, D. M. Grant, Anal. Chem. 1992, 64, 3133-3149. R. Dunkel et al, Anal. Chem. 1992, 64, 3150-3160.
11.3
Heteronuclear correlation through scalar coupling
a. X detected (not treated in the course, outdated) HETCOR R. Freeman, G. A. Morris, J. Chem. Soc. Chem. Commun. 1978, 684. G. Bodenhausen, R. Freeman, J. Magn. Reson. 1977, 28, 471-476. A. Bax, S. K. Sarkar, J. Magn. Reson. 1984, 60, 170-176. D. T. Pegg, M. R. Bendall, J. Magn. Reson. 1983, 55, 114-127. With homodecoupling of protons (BIRD): A. Bax, J. Magn. Reson. 1983, 53, 517-520. Long range HETCOR G. E. Martin, A. S. Zektzer, Magn. Reson. Chem. 1988, 26, 631-652. C. Wynants, K. Hallenga, G. van Binst, A. Michel, J. Zanen, J. Magn. Reson. 1984, 57, 93-98.
11.3
b. 1H detected methods ("inverse", "reverse") C. Griesinger, H. Schwalbe, J. Schleucher, M. Sattler, Two-Dimensional NMR Spectroscopy, Applications for Chemists and Biochemists, Chapter 3, Ed. W. R. Croasmun, R. M. K. Carlson, VCH Publishers, Weinheim, 2nd Edition, 1994, 457-580. Correlation through 1J XH Multiquantum spectroscopy in general: L. Mller, J. Am. Chem. Soc. 1979, 101 , 4481-4484. HMQC: A. Bax, R. H. Griffey, B. L. Hawkins, J. Am. Chem. Soc. 1983, 105 , 7188-7190. A. Bax, S. Subramanian, J. Magn. Reson. 1986, 67, 565-569. (BIRD filter) HSQC: G. Bodenhausen, D. J. Ruben, Chem. Phys. Lett. 1980, 69, 185-189. T. J. Norwood, J. Boyd, I. D. Campbell, FEBS Lett. 1989, 225, 369-371. Long range correlation: HMBC A. Bax, M. F. Summers, J. Am. Chem. Soc. 1986, 108 , 2093-2094. Gradient methods A. A. Maudsley, A. Wokaun, R. R. Ernst, Chem. Phys. Lett. 1978, 55, 9-14. R. E. Hurd, J. Magn. Reson. 1990, 87, 442. R. E. Hurd, B. K. John, J. Magn. Reson. 1991, 91, 648-653. A. L. Davis, E. D. Laue, J. Keeler, D. Moskau, J. Lohman, J. Magn. Reson. 1991, 94, 637-644. A. L. Davis, J. Keeler, E. D. Laue, D. Moskau, J. Magn. Reson. 1992, 98, 207-216. J. R. Tolman, J. Chung, J. H. Prestegard, J. Magn. Reson. 1992, 98, 462-467. J. Ruiz-Cabello, P. C. M. van Zijl, et. al, J. Magn. Reson. 1992, 100, 282. WATERGATE: M. Piotto, V. Saudek, V. Sklenr, J. Biomol. NMR 1992, 2, 661-665. Excitation sculpting: T.L. Hwang, A.J. Shaka, J. Magn. Reson. A 1995, 112, 275; K. Stot, J. Stzonehouse, T.L. Hwang, A.J. Shaka, J. Am. Chem. Soc 1995, 117, 4199-4200.
11.4
11.4
NOE
The classic book: J. H. Noggle, R. E. Schirmer, The Nuclear Overhauser Effect, Academic Press, New York, 1971. Best book on NOE (with many citations of primary literature): D. Neuhaus, M. Williamson, The Nuclear Overhauser Effect in Structural and Conformational Analysis, VCH Publishers, New York, 2nd Edition, 2000. 1D- NOE-Difference (steady state NOE): R. Richarz, K. Wthrich, J. Magn. Reson. 1978, 30, 147-150. G. E. Chapman, B. D. Abercrombie, P. D. Cary, E. M. Bradbury, J. Magn. Reson. 1978, 31, 459-469. TOE G. Wagner, K. Wthrich, J. Magn. Reson. 1979, 33, 675. Transient NOE A. A. Bothner-By, J. Spevacek, Pure Appl. Chem. 1982, 54, 569. NOESY S. Macura, R. R. Ernst, Mol. Phys. 1980, 41, 95-117. Artifacts in NOESY: S. Macura, K. Wthrich, R. R. Ernst, J. Magn. Reson. 1982, 46, 269-282. ROESY (CAMELSPIN) A. A. Bothner-By, R. L. Stephens, J-M. Lee, C. D. Warren, R. W. Jeanloz, J. Am. Chem. Soc. 1984,
106, 811.
A. Bax, D. G. Davis, J. Magn. Reson. 1985, 63, 207-213. Artifacts in ROESY-spectra: D. Neuhaus, J. Keeler, J. Magn. Reson. 1986, 68, 568-574. Improved versions: A. Bax, J. Magn. Reson. 1988, 77, 134-147. C. Griesinger, R. R. Ernst, J. Magn. Reson. 1987, 75, 261-271. J. Cavanagh, J. Keeler, J. Magn. Reson. 1988, 80, 186-194.
11.5
11.5
3J XH
Coupling constants and Karplus relationships X=13C or15N
J. Titman, D. Neuhaus, J. Keeler, J. Magn. Reson. 1989, 85, 111-131. H. Kessler, U. Anders, G. Gemmecker, J. Magn. Reson. 1988, 78, 382-388. W. Bermel, K. Wagner, C. Griesinger, J. Magn. Reson. 1989, 83, 223-232. H. Kessler, C. Griesinger, K. Wagner, J. Am. Chem. Soc. 1987, 109 , 6927-6933.
3J HP
D. Neuhaus, K. Wagner, M. Vasak, J. H. R. Kgi, K. Wthrich, Eur. J. Biochem. 1984, 143, 659. C. Griesinger, O. W. Srensen, R. R. Ernst, J. Chem. Phys. 1986, 85, 6387. V. Sklenar, A. Bax, J. Am. Chem. Soc. 1987, 109 , 7525-7526. Karplus-Relationships J. L. Marshall, Carbon-Carbon and Carbon-Proton NMR-Couplings: Applications to Organic Stereochemistry and Conformational Analysis, Verlag Chemie International, Florida, 1983. S. W. Homans, Oligosacharide Conformations: Applications of NMR and Energy Calculations, Prog. Nucl. Magn. Reson. Spectrosc. 1990, 22, 55-81. J. Marshall, D. E. Miller, S. A. Conn, R. Seiwell, A. M. Ihrig, Acc. Chem. Res. 1974, 7 , 333-339. V. F. Bystrov, A. S. Arseniev, Y. D. Gavrilov, J. Magn. Reson. 1978, 30, 151-184. M. T. Cung, M. Marraud, J. Neel, Macromolecules, 1974, 7, 606-613. P. E. Hansen, J. Feeney, G. C. K. Roberts, J. Magn. Reson. 1975, 17, 249-261. V. F. Bystrov, Progr. Nucl. Magn. Reson. Spectrosc. 1976, 10, 41-81. H-C-O-P: P. P. Lankhorst, C. A. G. Haasnoot, C. Erkelens, C. Altona, J. Biomol. Struct. and Dynam. 1984, 1, 1387-1405. W. Kung, R. E. Marsh, M. Kainosho, J. Am. Chem. Soc. 1977, 99, 5471-5477.
11.6
11.6 APT BB BIRD
Abbreviations and Acronyms used in this course Attached Proton Test Broadband Bilinear Rotation Decoupling Cross-relaxation Appropriate for Minimolecules Emulated by Locked Spins Composite-Pulse Decoupling Correlated Spectroscopy Continuous Wave Cyclically Ordered Phase Sequence Distortionless Enhancement by Polarization Transfer Double Quantum Coherence Double Quantum Filter Exclusive Correlation Spectroscopy Exchange Spectroscopy Fast Fourier Transform Fourier Transform Globally Optimized Alternating Phase Rectangular Pulse Gradient-Accelerated Spectroscopy
1H, 13C chemical-shift correlation spectroscopy
CAMELSPIN CPD COSY CW CYCLOPS DEPT DQC DQF E. COSY EXSY FFT FT GARP GRASP H,C-COSY HETCOR HMBC HMQC HOHAHA HSQC INADEQUATE INEPT INVERSE MLEV
Heteronuclear Correlation Spectroscopy Heteronuclear Multiple-Bond Correlation Heteronuclear Multiple Quantum Coherence Homonuclear Hartmann-Hahn Spectroscopy Heteronuclear Single Quantum Coherence Incredible Natural Abundance Double Quantum Transfer Experiment Insensitive Nuclei Enhanced by Polarization Transfer H, X correlation via 1H detection M. Levitt's CPD sequence
11.7
MQC MQF NOE NOESY RF ROESY SPT SQC TOCSY TOE TPPI TRNOE WALTZ WATERGATE ZQC T1 T2 tm c
Multiple Quantum Coherence Multiple Quantum Filter Nuclear Overhauser Effect Nuclear Overhauser Effect Spectroscopy Radio Frequency Rotating Frame Overhauser Effect Spectroscopy Selective Population Transfer Single Quantum Coherence Total Correlation Spectroscopy Truncated NOE Time-proportional Phase Incrementation Transient Nuclear Overhauser Effect CPD Sequence Water suppression pulse sequence Zero Quantum Coherence Longitudinal (spin-lattice) relaxation time for Mz Transverse (spin-spin) relaxation time for Mxy mixing time correlation time
11.8
11.7
How to describe the results and experimental conditions of 2D NMR spectroscopy in publications.
In contrast to 1D-NMR, there are no IUPAC recommendations for 2D spectroscopy yet. Some of the acronyms for individual methods are generally used and accepted (DQF.COSY, HSQC, HMBC, NOESY, ROESY), others are still used only by particular groups and may have synonyms (TOCSY and HOHAHA, CAMELSPIN e.g.). If possible, one should therefore always cite the original reference where the applied pulse sequence was first described. Do not use vendor specific acronyms or names. If a standard pulse sequence of the vendor's pulse sequence library was used, cite the application program package of the spectrometer manufacturer together with the name of the pulse sequence (e.g. DQF.COSY: standard pulse sequence cosydftp; BRUKER XWINNMR Version 2.6). Standard conditions to be listed: Solvent; sample concentration; for aqueous systems: pH and buffer concentration; temperature; field (in Tesla) or frequency (in MHz, for each nucleus separately). State whether the chemical shift reference was an external (phosphoric acid in a capillary for 31 P) or internal standard (TMS), or the solvent signal. Beware of using the solvent signal for reference with H2O/D2O; its chemical shift is very dependent on pH and temperature! The following acquisition and processing parameters should be indicated: Acquisition: In f1 (frequency domain corresponding to t1): Spectral width, maximal acquisition time (t1max = number of FIDs in t1 x t1-Increment) or Spectral width and number of increments in t1. Mode of quadrature detection in 1: TPPI or RSH. In f2: Spectral width, acquisition time or spectral width and number of acquired data points. Number of transients per FID , repetition time (time from the start of one pulse sequence to the next one) For H2O or D2O: indicate whether solvent was suppressed and with which method. For pulse sequences with spin lock (TOCSY, ROESY): B 1-filed in kHz (not in dB), Mode: e.g. MLEV17 DIPDY-2 or CW. For pulse sequences with mixing times (TOCSY, NOESY, ROESY): duration of the mixing in ms. For sequences with BB-decoupling: mode of composite pulse decoupling: WALTZ16, MLEV, GARP. Useful, but often omitted: phase cycles, number of dummy scans, with or without spinning.
11.9
Processing: For each dimension: zero filling, window function, phase sensitive or magnitude or power mode representation, baseline correction. Examples:
1H,31P-COSY, 1H-detected [1], 400/162 MHz, D O, 0.1 N phosphate buffer, pH=7.5, c=7.5 mM, 2
T=34. In F1: Spectral width 2000 Hz, 512 increments, TPPI. In F2: spectral width 5300 Hz, 2K data points, 32 scans/FID. Processed with zero filling to 1K in F1, cos2-filter in F1 and F2, phase sensitive. ROESY: 400 MHz, H2O/D2O 10:1, 0.1 N phosphate buffer, pH=7.5, c=7.5 mM, T=34. H2O suppression by presaturation. CW-spin lock (2.5 kHz) with flanking trim pulses (2 ms) [2], tm = 150 ms. In F1: 256 increments, TPPI. In F2: spectral width 5300 Hz, 2K data points, 32 scans/FID, Repetition rate 3.2 s. Processed after zero filling to 1K in F1, exp. line broadening by 3 Hz in F1, sine-filter shifted by /3 in F2. Presentation of results: The times when every 2D spectrum was worth a figure are long gone. As in 1D NMR, only particular sections showing information that is crucial to the conclusions are reproduced as graphics. In general, the information is best represented in tables giving the chemical shifts, coupling constants and assignments for both 1H and 13 C. It is a good practice to indicate by a footnote, whether an assignment is based on chemical shift arguments (and therefore speculative) or on a 2D correlation. For structure elucidation work, a formula of the final structure with arrows indicating observed correlations such as NOEs is often the easiest to read (as a supplement to the tables).

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40-042 Analytische Chemie IV

Structure Determination by NMR

Prof. Bernhard Jaun

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

What we would like to achieve with this course:

Subjects that are not part of this course:

Outline of the course:

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

Practical aspects of pulse Fourier transform NMR spectroscopy

1.1 The basic NMR experiment: physical description

The spin angular momentum J is associated with a magnetic moment as well:

The torque T acts on . In response,

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

Quantum mechanics: Quantum mechanical description of the spin angular momentum J : J = h I

: nuclear spin operator I

The Larmor (circular) frequency is:

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

sum over total sample volume inside the coil

For I = 1/2, > 0 (e.g. 1H):

1.2 Excitation by radio frequency pulses

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

Phase and amplitude of excitation

Phase and amplitude of excitation

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

Evolution of transverse magnetization under chemical shifts and scalar couplings

y My = My0 cos(t) >0

x Evolution of chemical shifts in the xy plane

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

1.3 Analog to digital conversion und Fourier transform

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

Folding after a real Fourier transform (cos-transform)

signal with 1 < N

Folding after a complex Fourier transform

signal with 1 < N

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

Complex analytical FT:

S(i) = {SA (kt) + iSB(kt)} exp(i2lk / N)

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

the instrumental line

) leads to Gaussian instead of

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

1.4 Quadrature detection

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

Illustration of the principle:

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

Redfield method of quadrature detection

SW* = 2 SW -> dwell = 1/ 2 SW

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

1.5 Phase cycles Cyclops

A-B-A+B = 4 x cos t channels A and B are fully equalized B+A-B-A = 4 x sin t

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

Disadvantage of phase cycles:

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

gradients tg in phase dephased

Advantages of gradients for coherence transfer pathway selection

Prof. B. Jaun: Structure determination by NMR / Analytische Chemie IV

1.6 Dynamic range and solvent suppression

+volt Receiver most significant bit

least significant bit