BASICS in NMR-Spektroskopie - Department für Chemie …€¦ · H. δ. H →Simplification of NMR Spectra and increase in . intensity of neighbouring. protons. NOe ~ 1/ r. 6. Distance

Introduction

Nuclear Magnetic Resonance, discovered 1946 Purcell & Bloch

EnergieEo Grundzustand

E1 Angeregter Zustand Absorption

Emission

Difference in energyE1 - E0 = h.ν

Frequency (Hz)ν = c / λ

BASICS in NMR-Spektroskopie

h...Planck‘sconstant6,63 . 10 -34 Js

I = 0 for nuclei of even mass- and atomic numbers, the even-even nucleie.g. 12

6C, 168O, 32

16S

Isotope spin I abundance (%) γ (gyromagnetic ratio)__________________________________________________

1H 1/2 99.98 26.82H 1 0.016 4.113C 1/2 1.108 6.719F 1/2 10031P 1/2 100 10.815N 1/2 0.37___________________________________________________

Nuclear Spin (I) and natural Abundance

Magnetic Moments and Energy Levels in the Magnetic Field

EnergieEo

E1 E = -μ . B0 μ = magnetic Moment= γ . I . h/2π

B0 = magnetic Density

Possible energy levels : 2 I + 1for I = 1/2

Energie

Eo + 1/2

E1 - 1/2

„parallel“

„antiparallel“

Energy Separations in the Magnetic Field

E = -μ . B0

B0 = magnetic Density

Energie

Eo + 1/2

E1 - 1/2 106

106 + 10

Energie

Eo -1/2

E1 +1/2

B0

Δ E

increasing difference in energy separations = increasing sensitivity

400 MHz

80 MHz

Transversal Magnetization

90 ° Pulse



Free Induction Decay (FID)

Free Induction Decay (FID) and

Fourier-Transformation

Decrease in trans-Versal Magnetization

Spin-Lattice and Spin-Spin Relaxation

FID - Timedomain

Frequency domain

Fourier transformation

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

B0 +

Chemical Shielding

ν Substanz - ν Referenz (Hz)δ = ----------------------------------

ν0 (MHz)

ppm, Parts per million, 10-6

B0

H-Atom

1s Orbital+

Blokal = B0 (1 - σ)σ = Shielding Constant

For Spectra Calibration:Tetramethylsilane (TMS), δ = 0Acetone, Acetonitrile.....

Chemical Shielding of various Substance Classes

Multiple Bond Anisotropy

Aromatic Ring Current

+

+

O

+

+

H HH R ++- - - --

-

H

+

+-

H

H

H

H

H

H

H

H

H

HH

HH

H

H

H

H

H

[18]-Annulen

8.9 ppm -1.8 ppm

B0

Multiplicity of Resonance Lines due to the Spin-Spin For n magnetic equivalent Nuclei (n+1) Lines

Doublett (d) 1:1

Spin-Spin Coupling

HH

Orientation of Nuclear Spins ppm

δ H

Kopplungskonstante J (Hz)

HH

H

Orientation of Nuclear Spins

ppm

δ H

Triplett (t) 1:2:1

Spin-Spin Coupling

H

HH

H

Orientation of Nuclear Spins

ppm

δ H

Quartett (q) 1:3:3:1

11 1

1 2 11 3 3 1

1 4 6 4 1

Pascals Rule only valid for first Order spectra

J / ν0 . δ < 0.1

bzw. δ H ≠ δ H

Intensity Distribution according to Pascals Triangle

Coupling Constant

Coupling Nuclei Bonds J (Hz) ____________________________________________________

Direkt 13C-H, =13CH 1 JCH 120-170Geminal H-C-H, =CH2 2 2JHH 0 - 20

Vicinal H-C-C-H 3 3JHH 3 - 20„Long-range“ H-C=C-C-H 4 4JHH < 5____________________________________________________

HH

H

Jtrans (11-19) > Jcis (5 -14)

H HH H

Long range coupling < 1 Hz

Dependence of the Coupling constant of the Dihedral Angle „Karplus Equation“

H

Konformationen (Newman-Projektion)

HH

H

H

H

Φ

gauche-Konformation Φ = 60 °

anti-Konformation Φ = 180 °

Pulse Scheme of a simple 1H Experiment

1H Ethylcrotonate

Homonuclear Decoupling

Homonuclear Decoupling Double Resonance: Nuclear Overhauser Enhancement (nOe)

HH

ppm

δ H δ HSpin-Decoupling by irradiating with anadditional frequency

HH

ppm

δ H δ H

→Simplification of NMR Spectra and increase in intensity of neighbouringprotons

NOe ~ 1/ r6

Distance < 2.5 A

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 exchangeof 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 zurStrukturaufklärung organischer Verbindungen . - Berlin [u.a.] :Springer , 2001 ISBN: 3-540-41877-6 kart. : DM 79.90

Breitmaier, Eberhard : Vom NMR-Spektrum zurStrukturformel organischer Verbindungen . - Weinheim : WILEY-VCH , 2005ISBN: 3-527-31499-7 Pb. : EUR 39.90, sfr 64.00

Breitmaier, Eberhard : Structure elucidationby 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 99.98 26.813C 1/2 1.108 6.7

C13 OHH

H

HH

H

ppm

δ 13C

JC,H

C13 OHH

H

HH

H

ppm

δ 13C

Broad band decoupling→ Increase in intensity(NOe!)

13C NMR Spectra of Ethanol (Proton decoupled in CDCl3)

CDCl3

OCH2

CH3

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:

13.7 22.8 31.9 31.9 22.8 13.7

H3C--CH2--CH2--CH2--CH2--CH3

n -Hexanol: γ β α

14.2 22.8 32.0 25.8 32.8 61.9

H3C--CH2--CH2--CH2--CH2--CH2-OH

3 -Hexanol:

14.0 19.4 39.4 72.3 30.3 9.9

OH|

H3C--CH2--CH2--CH--CH2--CH3

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



13P NMR Spectroscopy

O

HO OH

O P

OO

OO

P

OO

__

NH2

N

N

N

N

O

P

OO

__

γ β α

Isotope I abundance (%) γ__________________________1H 1/2 99.98 26.831P 1/2 100 10.8

0 -10 -20 ppm

adenosintriphosphate

2D NMR SpectroscopyThe H-H- Correlated Spectroscopy (COSY)

Ethylcrotonate

2D NMR SpectroscopyThe H-H- (COSY)



staggered contourDisplay

2D NMR SpectroscopyThe H-H- COSY on Ethylcrotonate

2D NMR SpectroscopyThe long-range- COSY on Ethylcrotonate

32

54

2D NMR Spectroscopythe J-resolved COSY

2D NMR Spectroscopythe total correlated Spectroscopy (TOCSY)homonuclear Hartman-Hahn Echo HOHAHA

preparation

evolution

spinlockdetection

2D NMR Spectroscopythe total correlated Spectroscopy(TOCSY)homonuclear Hartman-Hahn Echo HOHAHA

strychnine

2D NMR Spectroscopythe nuclearoverhauser Spectroscopy NOESY

strychnine

2D NMR SpectroscopyThe hetero-nuclear multiple quantum coherenceSpectroscopy HMQC

Ethylcrotonate

2D NMR SpectroscopyThe hetero-nuclear single quantum coherenceSpectroscopy HSQC

Ethylcrotonate

2D NMR SpectroscopyJ-resolved 13C NMR

Ethylcrotonate

3D NMR Spectroscopythe third dimension

3D NMR SpectroscopyH-C-P Correlationon Triphenyl-phosphane

3D NMR SpectroscopyH,H,C-Correlationon 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 internucleardistance, 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 IntroductionThe 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 conditionsb) 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 ApplicationUpper 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 DMPC-d54/cholesterol

HR MAS ApplicationEffect of spinning speed at 400MHz on plant leaf proton-spectrum

HR MAS Application

More applications; Fruitsanalysis of the ripening processespenetration of chemicals: insectizides, herbizidesanalysis of changes in parts damaged by transport etc.investigation of the rot processes

HR MAS Application

HR MAS Application

BASICS in NMR-Spektroskopie - Department für Chemie …€¦ · H. δ. H →Simplification of NMR Spectra and increase in . intensity of neighbouring. protons. NOe ~ 1/ r. 6. Distance

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