BASICS in NMR-Spektroskopie Introduction Nuclear Magnetic Resonance, discovered 1946 Purcell & Bloch
E1
Angeregter Zustand
Absorption
Energie Eo
Difference in energy E1 - E0 = h.ν
Grundzustand
h...Planck‘s constant 6,63 . 10 -34 Js
Emission
Frequency (Hz) ν =c/λ
Nuclear Spin (I) and natural Abundance
I = 0 for nuclei of even mass- and atomic numbers, the even-even nuclei e.g. 126C, 168O,
32 S 16
Isotope spin I abundance (%) γ (gyromagnetic ratio) __________________________________________________ 1H
1/2 99.98 26.8 2H 1 0.016 4.1 13C 1/2 1.108 6.7 19F 1/2 100 31P 1/2 100 10.8 15N 1/2 0.37 ___________________________________________________
Magnetic Moments and Energy Levels in the Magnetic Field
E1
E = -μ . B0
μ = magnetic Moment = γ . I . h/2π
Energie Eo
B0 = magnetic Density Possible energy levels : 2 I + 1 for I = 1/2
E1 - 1/2
„antiparallel“
Eo + 1/2
„parallel“
Energie
Energy Separations in the Magnetic Field E = -μ . B0
400 MHz E1 - 1/2
106
B0 = magnetic Density
Energie Eo + 1/2
Eo -1/2
106 + 10
ΔE
Energie
E1 +1/2
80 MHz
B0
increasing difference in energy separations = increasing sensitivity
Transversal Magnetization
90 ° Pulse
Transversal Magnetization
Transversal Magnetization
Free Induction Decay (FID)
Free Induction Decay (FID) and Fourier-Transformation Decrease in transVersal Magnetization Spin-Lattice and SpinSpin Relaxation
FID - Timedomain Fourier transformation
Frequency domain
Prinziples of a superconducting magnet
1H
NMR Spectrum of Ethylalcohol
NMR-Parameters are Integral : relative amount of H-Atoms
Chem. Shift : Elektronic Environment
Spin-Spin Coupling : Sphärical Environment
Deep field shift
High field Shift
Chemical Shielding H-Atom
B0
δ
+
1s Orbital
ν Substanz - ν Referenz (Hz) = ---------------------------------ν0 (MHz)
ppm, Parts per million, 10-6
B0
+
Blokal = B0 (1 - σ) σ = Shielding Constant
For Spectra Calibration: Tetramethylsilane (TMS), δ = 0 Acetone, Acetonitrile.....
Chemical Shielding of various Substance Classes
Multiple Bond Anisotropy +
-
+
-
-
H
-
O
H
+
R
H
-
+
+ B0
H
H
+ H H
-
+
H
H
H H
8.9 ppm
H
H
-1.8 ppm H
H H
H
H
H
H H
H
[18]-Annulen
Aromatic Ring Current
+
Spin-Spin Coupling H
Multiplicity of Resonance Lines due to the Spin-Spin For n magnetic equivalent Nuclei (n+1) Lines
H
Kopplungskonstante J (Hz)
Orientation of Nuclear Spins
ppm
δH
Doublett (d) 1:1 H H H ppm
Orientation of Nuclear Spins
δH
Triplett (t) 1:2:1
Spin-Spin Coupling H H H H ppm
δH
Orientation of Nuclear Spins
Quartett (q) 1:3:3:1
Intensity Distribution according to Pascals Triangle 1 1 1 1 1 4
J / ν0 . δ < 0.1
1 2
3
1 3
6
Pascals Rule only valid for first Order spectra
bzw. δ H ≠ δ H
1 4
1
Coupling Constant Coupling Nuclei Bonds J (Hz) ____________________________________________________ 13C-H,
=13CH H-C-H, =CH2
Direkt Geminal
1 2
JCH 120-170 2J HH 0 - 20
3J Vicinal H-C-C-H 3 HH 3 - 20 4J „Long-range“ H-C=C-C-H 4 HH < 5 ____________________________________________________
H
H H
H
H
H
H
Jtrans (11-19) > Jcis (5 -14)
Long range coupling < 1 Hz
Dependence of the Coupling constant of the Dihedral Angle „Karplus Equation“ H Konformationen (Newman-Projektion) H
H
Φ
H H
gauche-Konformation Φ = 60 °
H
anti-Konformation Φ = 180 °
Pulse Scheme of a simple 1H Experiment
1H
Ethylcrotonate
Homonuclear Decoupling
Homonuclear Decoupling Double Resonance: Nuclear Overhauser Enhancement (nOe) H H
ppm
Spin-Decoupling by irradiating with an additional frequency
δH
δH
→Simplification of NMR Spectra and increase in intensity of neighbouring protons
H H
ppm
NOe ~ 1/ r6 Distance < 2.5 A
δH
δH
Homonuclear Decoupling of ethylcrotonate
Homonuclear Decoupling of Strychnin
Water suppression by presaturation
Water suppression of a sucrose sample in D2O
Signal separation using Eu(fod)3
Signal separation of enantiomers using a chiral shift reagent
Signal separation of enantiomers using a chiral solvating agent
Hydrogen/Deuterium exchange of Glycerol in DMSO
Low temperature calibration using methanol
Low temperature calibration using methanol
Dynamic 1H NMR spectroscopy with N,N-Dimethylformamide
1D NOE Spectroscopy
Quantitative 1H NMR Spectroscopy: Determination of the Alcohol Content
Symmetry and Spectra
Equipment
Bruker DPX 300 MHz
Equipment
Bruker Avance II 400 MHz
Literatur
Pretsch, Ernö : Spektroskopische Daten zur Strukturaufklärung organischer Verbindungen . - Berlin [u.a.] : Springer , 2001 ISBN: 3-540-41877-6 kart. : DM 79.90 Breitmaier, Eberhard : Vom NMR-Spektrum zur Strukturformel organischer Verbindungen . - Weinheim : WILEY-VCH , 2005 ISBN: 3-527-31499-7 Pb. : EUR 39.90, sfr 64.00 Breitmaier, Eberhard : Structure elucidation by NMR in organic chemistry . - Chichester [u.a.] : Wiley , 2004 ISBN: 978-0-470-85007-7
13C
NMR Spectroscopy
Isotope I nat. Abundance (%) γ ______________________________ 1H
1/2 1/2
13C
99.98 1.108
26.8 6.7
H H
13
H
C
OH
H
H
H H
JC,H
C H
ppm
δ 13C
H
13
OH H
Broad band decoupling → Increase in intensity (NOe!)
ppm
δ 13C
13C
NMR Spectra of Ethanol (Proton decoupled in CDCl3) CH3 OCH2
CDCl3
13C NMR Spectra of cholesteryl acetate, fully proton coupled
13C NMR Spectra of cholesteryl acetate, proton decoupled
13C NMR Spectra of cholesteryl acetate
13C
NMR Data (ppm) of Hydrocarbons / Alcohols
n -Hexane: H3C--CH2--CH2--CH2--CH2--CH3 13.7 22.8 31.9 31.9 22.8 13.7
n -Hexanol:
γ
β
α
H3C--CH2--CH2--CH2--CH2--CH2-OH 14.2 22.8 32.0 25.8 32.8 61.9
3 -Hexanol: OH | H3C--CH2--CH2--CH--CH2--CH3 14.0 19.4 39.4 72.3 30.3 9.9
Chemical Shifts of various functional groups
Composite Pulse Decoupling CPD
13C NMR of Ethylcrotonate
13C NMR of Glucose
Quantitative 13C NMR using Inverse Gated Decoupling Techniques
Quantitative 13C NMR using Inverse Gated Decoupling Techniques
13C NMR – Gated Decoupled Technique for C-H Couplings
13C NMR – Attached Proton Test (APT)
cholesterylacetate
13C NMR – pKa Determination of Ascorbic Acid
Ascorbic Acid Equilibrium
13C NMR – pKa Determination of Ascorbic Acid
13C NMR – pKa Determination of Ascorbic Acid
13P
NMR Spectroscopy NH2
Isotope I abundance (%) γ __________________________ 1H 1/2 99.98 26.8 31P 1/2 100 10.8
N
N _O
O_ P O
P
O
O
γ
_ O
O_ O
P
N
N O
O
O β
α
HO OH
adenosintriphosphate
0
-10
-20 ppm
2D NMR Spectroscopy The H-H- Correlated Spectroscopy (COSY) Ethylcrotonate
2D NMR Spectroscopy The H-H- (COSY)
2D NMR Spectroscopy The H-H- (COSY)
2D NMR Spectroscopy The H-H- (COSY)
staggered
Display
contour
2D NMR Spectroscopy The H-H- COSY on Ethylcrotonate
2D NMR Spectroscopy The long-range- COSY on Ethylcrotonate 2 3 4 5
2D NMR Spectroscopy the J-resolved COSY
2D NMR Spectroscopy the total correlated Spectroscopy (TOCSY) homonuclear Hartman-Hahn Echo HOHAHA preparation spinlock
evolution
detection
2D NMR Spectroscopy the total correlated Spectroscopy (TOCSY) homonuclear Hartman-Hahn Echo HOHAHA
strychnine
2D NMR Spectroscopy the nuclear overhauser Spectroscopy NOESY
strychnine
2D NMR Spectroscopy The heteronuclear multiple quantum coherence Spectroscopy HMQC
Ethylcrotonate
2D NMR Spectroscopy The heteronuclear single quantum coherence Spectroscopy HSQC
Ethylcrotonate
2D NMR Spectroscopy J-resolved 13C NMR
Ethylcrotonate
3D NMR Spectroscopy the third dimension
3D NMR Spectroscopy H-C-P Correlation on Triphenylphosphane
3D NMR Spectroscopy H,H,C-Correlation on Strychnine
HR-MAS High Reslolution Magic Angle Spinning
The line width of an NMR resonance depends strongly on the microscopic environment of the nucleus under study. Interactions such as the chemical shift and dipole-dipole coupling between neighboring spins are anisotropic and impose a dependence on the NMR frequency based on the orientation of a spin or molecule with respect to the main magnetic field direction. Furthermore, the magnetic susceptibility of the sample and susceptibility differences within the sample lead to broadening of the resonances.
HR-MAS
Introduction
In liquid state the rapid isotropic motion of the molecules averages the anisotropic interactions, resulting in an isotropic chemical shift frequency and a disappearance of the line broadening due to dipolar couplings. Furthermore the sample geometry, a cyclinder parallel to the main magnetic field, is chosen such that the susceptibility broadening is minimized.
HR-MAS Introduction
In solids on the other hand, the lack of molecular mobility results in broad lines. Because the magnitude of the coupling between two nuclear spins depends on the internuclear distance, the dipolar coupling is a through-space interaction. In contrast, J coupling requires the presence of chemical bonds. It is transferred through the electrons engaged in these bonds and thus is confined to nuclei within a molecule. Through-space dipolar coupling, however, also occurs between nuclei in different molecules.
HR-MAS Introduction
The two coupling mechanisms are therefore complementary in information content.
Three properties of the heteronuclear dipolar coupling Hamiltonian stand out: 1) The magnitude of the coupling is proportional to the product of the gyromagnetic ratios. 2) The dipolar coupling is inversely proportional to the cube of the inter-nuclear distance, so the interaction falls off rapidly as the nuclei are moved farther apart.
HR-MAS Introduction
3) The dipolar coupling is dependent on the orientation. This means that for two nuclei of spins I and S which are separated by a fixed distance, the magnitude of the dipolar interaction will be greater for certain orientations of the I ± S internuclear vector than for others.
HR-MAS Introduction
In a static solid sample comprised of randomly oriented crystallites, however, the internuclear vector remains invariant over time, and the resonance frequency produced by each crystallite depends on its orientation with respect to the external field. In a polycrystalline powder sample in which the crystallites are oriented in all possible directions, the presence of a heteronuclear dipolar coupling produces a spectrum such as that shown next;
HR-MAS Introduction
Proton spectra of a human Lipoma tissue. The top spectrum is acquired in a conventional high resolution probe (spinning at 20 Hz), while the lower spectrum is acquired in a HR-MAS probe (spinning at 5 kHz)
HR-MAS Introduction Dipolar pattern for two coupled spins in a powder sample; the two signals correspond to a positive (parallel spins) and a negative (antiparallel spins) value.
-d
-d/2
0
d/2
d
The points with maximum intensity corresponds to Φ 90–a perpendicular orientation to B0 is adopted by a majority of the crystals. At the magic angle of Φ 54.7o the dipolar coupling is zero.
HR-MAS Introduction The line broadening can be reduced by spinning the sample rapidly around an axis which is oriented at an angle Φ 54.7o with the direction of the magnetic field. By spinning at this so-called Magic Angle, at a rate larger than the anisotropic interactions, these interactions are averaged to their isotropic value, resulting in substantial line narrowing. The proton spectra of HMW (high molecular weight) subunits of wheat in D2O a) Under static conditions b) Obtained with a HR-MAS probe at 10 kHz spinning rate
HR-MAS Introduction In addition to pure solids or pure liquids there is a wide range of materials which exhibit either reduced or anisotropic mobility. Examples include polymer gels, lipids, tissue samples, swollen resins, plant and food samples. While these samples generally have sufficient mobility to greatly average anisotropic interactions, the spectral resolution for the static samples is still much lower than that which achieved for liquid samples.
HR-MAS Introduction The excess broadening under static conditions is due to a combination of residual dipolar interactions and variations in the bulk magnetic susceptibility. For a variety of samples, including the aforementioned examples, magic angle spinning is efficient at averaging this left-over components of the solid state line width and leads to NMR spectra that display resolution approaching that of liquid samples. Such methods have been termed HIGH Resolution MAS NMR
HR MAS Application
HR-MAS Application Upper probe chamber with MAS stator in magic angle position. (pneumatic switch) MAS pneumatic control unit for sample spinning (up to 17 kHz)
HR-MAS Application
HR-MAS Application
A magnifying glass makes life easier
HR-MAS Application HR-MAS Application
HR-MAS Application
HR-MAS Application
23Na,
sodium phosphate
HR-MAS Application
HR MAS Application Detection of natural abundance 1H–13C correlations of cholesterol in its membrane environment
Dependence on the spinning frequency the 1H MAS NMR spectrum of a sample of multilamellar vesicles of DMPCd54/cholesterol
HR MAS Application Effect of spinning speed at 400MHz on plant leaf proton-spectrum
HR MAS Application More applications; Fruits analysis of the ripening processes penetration of chemicals: insectizides, herbizides analysis of changes in parts damaged by transport etc. investigation of the rot processes
HR MAS Application
HR MAS Application
