Transcript Lecture 15

Relaxation Agents (Claridge 4.1)
Contact Shifts/pseudo contact shifts Gunther 10.6
Solvent Suppression (Claridge 10.5-10.6)
Diffusion NMR: Claridge 9
Solvent Exchangeable Protons
Protons such as OH and NH exchange with water, affecting their detection by NMR.
Depending upon the exchange rate and the chemical shift difference between the water
line and the resonance, the resonance could be broadened, possibly to the point of not
being observed.
How to identify solvent exchangeable resonances
1) Broad resonance
2) Temperature Dependence of Chemical Shift
3) Solvent effects on chemical shift
Resonances of exchangeable protons will shift with water according to the
chemical shift of water in various solvents (see table)
4) D2O Exchange
5) Presaturation
6) ROESY/NOESY/1D ROE
How to observe exchangeable protons better
1) Remove as much water out of the solvent as possible
2) Lower temperature
3) Adjust pH
4) Change magnetic field
5) Change solvent
Solvent Suppression
Solvent suppression:
A general term for several techniques used to eliminate or nearly eliminate large
(intense) resonances
Large resonances obscure smaller resonances underneath the large peak
Large resonances dominate the fid so much that the difference between it and
every other resonance is so great that it occupies the whole space of the ADC.
Solvent Suppression
Why would you want to suppress solvent or why would you just not use
deuterated solvent?
1) Exchangeable Protons
Some protons exchange with H2O, and would become deuterated in D2O (or
CD3OD etc.).
2) Cost
3) Ease
4) Cannot dry sample, dissolve in deuterated solvent
Solvent Suppression
What are the drawbacks of using solvent suppression rather than
deuterated solvents?
1) No Lock Signal
2) Hard to Shim
3) Hard to Suppress Multiple Resonances
4) Phasing
5) Obscured Resonances
Techniques to Suppress Solvent
1) Jump-return or binomial pulse (11, 1331, etc.)
2) Presaturation
3) Gradient Enhanced Solvent Suppression (WATERGATE, WET)The best way to suppress solvent signals is with pulsed field gradients to be
certain that no solvent signal remains observable prior to acquisition.
PFGs work on the transverse magnetization (in the x-y plane).
The two most common are WATERGATE (WATER suppression by GradientTailored Excitation) and WET (Water Eliminated through Transverse Gradients).
Both WATERGATE and WET can be added to most multidimensional
experiments as well as the basic 1D (so there are WATERGATE NOESY, WET
NOESY, WET-DQ-COSY, etc.).
The WATERGATE uses a gradient spin-echo G1-S-G1 sequence where G1 is a
PFG and S is a series of pulses (such as the binomial or jump-return sequence)
that creates no net effect on the solvent resonance but a 180º rotation of
everything else.
Shaped Soft Pulses
The most straightforward way to excite only a specific region on the spectrum is with soft
pulses.
Soft pulses are pulses with a low B1 field, long pulses at low power (as in presaturation).
Ideal pulses would excite everything within the proper region and nothing outside of it.
A standard pulse is a rectangular pulse.
The Fourier transform of a rectangular pulse is a sinc(x) function that we discussed earlier.
The sinc pulse is a problem because of the damped oscillation of excitation.
Similar to what was discussed with apodization, a better option would be one that does not
oscillate.
The most simple solution is to use a Gaussian pulse.
Many other shapes of pulses have been applied for specific purposes.
Shaped Soft Pulses
Gaussian Pulse with an array of transmitter offsets
Rectangular (Sinc) Pulse with an array of transmitter offsets
Solvent Suppression during Processing
Resonances at a given frequency can be suppressed through data processing techniques,
usually digital filtering.
The most common is by filtering out the on-resonance component of the FID (the solvent)
with a digital filter that selects the on-resonance component and then subtracts this from the
FID (so the peak at the center of the spectrum).
Additional filtering can be done by fitting the FID to a polynomial function and subtracting
that from the FID.
2 mM Sucrose, 90% H2O
Watergate with solvent
supression during processing
without solvent supression
during processing
1D of peptide in 95% H2O
2D ROESY in 95% H2O
Demo, presat class project sample, wet protein
NMR of Paramagnetics
Paramagnetic materials will cause one or more of the following in
NMR spectra:
1) Shortening of T1
(relaxation agents)
2) Broadening of NMR lines (shortening of T2)
(broadening agents)
3) Contact Shifts
4) Pseudocontact shifts
(shift reagents)
Relaxation Agents
Electrons have spin 1/2 and a gyromagnetic ratio 658 times that of a proton, and
thus are very efficient at inducing intermolecular dipole-dipole relaxation (T1).
Even low concentrations of paramagnetic species can severely reduce relaxation
rate, which eliminates NOE enchancement.
Reducing relaxation rate (at least to a certain point) can be good.
Since T1 times are sometimes seconds to hundreds of seconds (quarternary
carbons), it is unreasonable to wait that long between scans.
Using shorter pulse widths is one way around this problem (recall the discussion
of repetition rate and the Ernst angle); an alternative is adding paramagnetic
species
If paramagnetic species are added for this purpose, they are relaxation agents.
Relaxation Agents
All paramagnetic species are not good relaxation agents.
The ideal paramagnetic ion to use is one that acts on the whole molecule,
reducing T1 without severe line broadening and without significant change in
chemical shifts
The molecule most commonly used as a relaxation agent is
chromiumacetylacetonate (Cr(acac)3) or simply chromium acac.
Loss of heteronuclear NOE
C13 peak integration
Significant problems to the addition of relaxation reagents include: effects on
the sample (degradation, precipitation, etc.), solubility of the relaxation agent,
and difficulty controlling the amount of T1 reduction.
Shorten the relaxation time T1
13C
Spectrum of camphor
With Cr(acetyl acetonate)
No Cr(acetyl acetonate)
Broadening agents/Paramagnetic Relaxation
Enhancement Agents (PREs)
Paramagnetic material with long electron relaxation times induce large variation in
nuclear relaxation times without affecting chemical shift much.
These also normally have little effect on general T1 times, and are usually site
specific
To use properly, you need to have an idea of the concentration range (nM- M)
where the shortening of T1/T2 is local; that way, there is broadening of resonances
specifically near where the paramagnetic agent is interacting.
Included in paramagnetics with long electron relaxation times: Mn(II), Cu(II), and
Gd(III).
Broadening agents
Titration of Mn (II) into RNA sample (~2 mM). Imino portion of 1D spectrum of RNA;
several iminos broaden before others, indicating metal ion binding site.
Contact Shifts
Paramagnetic compounds such as radical anions of aromatic hydrocarbons can lead to
Fermi contact shifts.
This interaction leads to a hyperfine splitting of the electron signal in electron spin
resonance spectroscopy (ESR), while shifting the NMR signal of the ligand to higher
or lower field.
a = Q
a = the hyperfine coupling constant for the scalar interaction between the electron and
nuclear spin,
Q = proportionality constant (in Gauss)
 = unpaired spin density at the atom under consideration.
The hyperfine coupling constant is essentially the same as the scalar coupling constant
(J) of the NMR signal.
Contact Shifts
The signal of the unpaired electron is split by magnetic nuclei within the radical.
If the paramagnetic compound is in low concentration relative to the diamagnetic
compound, the paramagnetic spin density can be spread over the large number of
molecules- it is diamagnetically diluted.
If electron spin relaxation is fast, then the NMR lines are observable.
Spin-spin coupling is effectively removed because the electron relaxation is fast and
the exchange of electrons cause the diamagnetic compound to see an average.
You might expect to see an average NMR signal, essentially independent of the
paramagnetic ion- that is not what is observed, as there is a contact shift.
Since the energy difference of the ESR signal is much different than the NMR signal,
there is a population difference and the one state is favored in the time averaged
signal.
Contact Shifts
B/B0 = ae2/(4pkT)
B = the contact shift
B0 = magnetic field
a = the hyperfine splitting
e = gyromagnetic ratio of the electron
p = gyromagnetic ratio of the proton
Additionally, the line width is proportional to the hyperfine coupling constant, and 1/r6
where r is the distance between the nucleus and the radical center.
1-propylnapthalene in the
presence in varying
concentrations of radical anion
Pseudocontact Shifts (Shift Reagents)
Shift reagents are pseudocontact shifts of proton resonances induced by a strongly
anisotropic paramagnetic center, such as the unpaired electrons in the valence orbitals
of rare earth metals [also low spin iron (III) and Cobalt (II) normally].
This is a dipolar interaction between the paramagnetic center and the nucleus through
space.
The magnitude of the dipolar interaction is proportional to:
(3cos2 - 1)/r3
r = distance between the paramagnetic center and the nucleus
= angle between the effective symmetry axis of the paramagnetic moment and the
distance vector to the nucleus.
Pseudocontact Shifts
Lanthanides are often used as shift reagents, particularly europium, because they
induce only slight line broadening.
Primary use of shift reagents is to spread complicated spectral regions over a much
larger chemical shift range.
The pseudocontact shift results from complex formation between the shift reagent,
where the reagent has free coordination sites, and the substrate.
There is structural information based upon the amount of shift.
 = *(3cos2 - 1)/r3
 = empirical constant for the complex being studied.
 = shift
r = distance between the paramagnetic center and the nucleus
2-adamantanol (with and
without EuIII complex)
Chiral Shift Reagents
NMR cannot distinguish enantiomers.
However, by forming diastereomers by reaction with an enantiomer pure substance,
the diastereomers are distinguishable by NMR.
The reaction can be in fast equilibrium, as NMR will detect the average chemical
shift value.
In fast equilibrium, the line width is proportional to the frequency difference
between the two resonances.
Lanthanides can induce very large shifts causing the resonance to broaden
significantly.
~ ()2/2k
 = line width
 = frequency difference (in Hz)
k = exchange rate
Chiral Shift Reagents
Since the frequency difference in Hz is correlated to the magnetic field
 ~ B02(exp(G/RT))
Thus, the lines are broader on high field instruments as the line width is
proportional to the square of the applied field.
However, the lines will be narrower at high temperature as the line width is
inversely proportional to absolute temperature.
Ideally, use the lowest field instrument at the highest temperature possible.
1H/13C NMR on Paramagnetic Compounds
Pascal Roquette; Astrid Maronna; Matthias Reinmuth; Elisabeth Kaifer; Markus Enders; Hans-Jörg
Himmel; Inorg. Chem. 2011, 50, 1942-1955
Désirée C. Sauer; Matthias Kruck; Hubert Wadepohl; Markus Enders; Lutz H. Gade;
Organometallics 2013, 32, 885-892.
Désirée C. Sauer; Matthias Kruck; Hubert Wadepohl; Markus Enders; Lutz H. Gade; Organometallics 2013, 32, 885-892.
Contact and Pseudocontact Shifts in Fe-S Proteins
Magnetic Susceptibility
Evans Method
1D spectrum- measure shift from paramagnetic
Sample in 5mm tube, paramagnetic solution in capillary
(0 – i)/o = (-4/3)( Ci – Co)
o = Frequency of Resonance in outer tube
i = Frequency of Resonance in inner tube
C = Volume susceptibility
Diffusion NMR
Diffusion NMR or Diffusion Ordered Spectroscopy (DOSY) is the study of
molecular diffusion in solution which is correlated to molecular size, shape, and
aggregation state.
Diffusion NMR can be used to study molecular diffusion or be used as a basis for
separating out NMR spectra of molecules in a mixture.
The study of diffusion rates depends upon application of pulsed field gradients.
The gradients are used to identify the physical location of a molecule in solution.
The molecular diffusion is then analyzed in the direction of the gradient (most
likely the Z-axis, but could be X or Y-axis).
The sequence applied is a PFG spin-echo; the first pulse rotates the magnetization
from the Z-axis to the Y-axis and the chemical shift evolves in the X-Y plane for
time .
Without the gradient pulse, the chemical shift information will be refocused by the
180º pulse, and another time  period as in a standard spin-echo experiment.
The only intensity change of the resonances should be due to T2 relaxation.
If the gradient pulses are added into the sequence, complete refocusing of the
chemical shift will only occur if the molecule has not moved, as the gradients will
selectively defocus and refocus magnetization according to position in the magnet.
If a molecule moves (diffuses), then the intensity of the resonance detected will be
weaker depending how far it moved.
The amount the molecule moved will be dependent upon its diffusion coefficient
and the length of the time it is allowed to move for.
The intensity detected is defined by:
I = I0 exp(-2/T2 - (G)2D( - /3))
I0 is the signal intensity without
gradients (or with power of gradients =
0)
G is the gradient strength
D is the Diffusion coefficient
 and  are the experimental delays in
the pulse sequence
Plotting ln(I/I0) vs. G2 will yield a linear
plot with a slope proportional to the
Diffusion coefficient.
PFG Spin-Echo of mixture of Acetone, CDCl3, TMS, Isomenthol
Temp = 298K,  = 30 ms, gradient strengths were increased from 0.025 T/m to 0.25
T/m
ln(I/I0) vs G2
The slopes indicate relative mobility rates of 1:1.3:1.5:1.6
(isomenthol:TMS:Acetone:CHCl3)
Signal intensity at increasing gradient strength
Plot of ln(I/I0) vs. Gradient2
Diffusion Study
Sturlaugson, Fayer, et al., J.Phys.Chem (2010)
Diffusion Study of Ion Pairing
Solvent dependence of the diffusion coefficient D (10−10 m2 s−1)
and hydrodynamic radius rH (Å) of representative Au(I) complexes
Complex
[IPrAu(NCPh)]BAr4F
CD2Cl2
[IPrAu(NCMe)]SbF6
Solvent
CDCl3 Cation
Anion
Cation
Anion
CDCl3 Cation
CD2Cl2 Cation
D
6.0
5.9
8.7
8.7
7.0
10.3
rH
7.2
7.3
6.6
6.6
6.3
5.8
Diffusion coefficients (D) obtained from PFGSE data with Stejskal-Tanner plots.
Hydrodynamic radii (rH) calculated from Stokes-Einstein equation
Lau, Gorin, and Kanan, Chemical Science, 5: 4975-4979 (2014)
DOSY
DOSY is a 2D experiment sort of; there is no t1, but the results are plotted as a 2D.
Plot of chemical shift vs. Diffusion coefficient
Used to identify compounds in a mixture based upon chemical shift and diffusion
coefficient
The relative intensities of the resonances can be extracted out, and then identifying
the ratio of compounds in the mixture becomes possible.
Real 2D sequences can be incorporated to create a 3D sort of.
The pseudo 3D can have one dimension of 13C one of 1H and one of diffusion
coefficient (or you could have a Diffusion Coefficient Edited COSY, so a 3D COSY
...)]
DOSY
Diffusion Measurement of Polymer
Polydispersity index of polymers revealed by DOSY NMR, Justine Viéville a, Matthieu Tanty, Marc-André Delsuc, Journal of Magnetic Resonance 212 (2011) 169–173
Diffusion Measurement of Polymer
Polydispersity index of polymers revealed by DOSY NMR, Justine Viéville a, Matthieu Tanty, Marc-André Delsuc, Journal of Magnetic Resonance 212 (2011) 169–173