NMR Spectroscopy Part II. Signals of NMR

Free Induction Decay (FID) FID represents the time-domain response of the spin system following application of an radio-frequency pulse. With one magnetization at  , receiver coil would see exponentially decaying signal. This decay is due to relaxation.

Fourier Transform The Fourier transform relates the time-domain f(t) data with the frequency-domain f(w) data.

Fourier Transform

NMR line shape Lorentzian line Aamplitude Whalf-line width

Resolution Definition For signals in frequency domain it is the deviation of the peak line-shape from standard Lorentzian peak. For time domain signal, it is the deviation of FID from exponential decay. Resolution of NMR peaks is represented by the half-height width in Hz.

Resolution

Resolution-digital resolution

Resolution Measurement half-height width: 10~15% solution of 0-dichlorobenzene (ODCB) in acetone Line-shape: Chloroform in acetone

Resolution Factors affect resolution Relaxation process of the observed nucleus Stability of B 0 (shimming and deuterium locking ) Probe ( sample coil should be very close to the sample ) Sample properties and its conditions

Sensitivity Definition signal to noise-ratio A : height of the chosen peak N pp :peak to peak noise

Sensitivity Measurement 1 H 0.1% ethyl benzene in deuterochloroform 13 C ASTM, mixture of 60% by volume deuterobenzene and dioxan or 10% ethyl benzene in chloroform 31 P 1% trimehylphosphite in deuterobenzene 15 N 90% dimethylformamide in deutero-dimethyl- sulphoxide 19 F 0.1% trifluoroethanol in deuteroacetone 2 H, 17 O tap water

Sensitivity Factors affect sensitivity Probe: tuning, matching, size Dynamic range and ADC resolution Solubility of the sample in the chosen solvent

Spectral Parameters Chemical Shift Caused by the magnetic shielding of the nuclei by their surroundings. d-values give the position of the signal relative to a reference compound signal. Spin-spin Coupling The interaction between neighboring nuclear dipoles leads to a fine structure. The strength of this interaction is defined as spin- spin coupling constant J. Intensity of the signal

Chemical Shift Origin of chemical shift   shielding constant  Chemically non-equivalent nuclei are shielded to different extents and give separate resonance signals in the spectrum

Chemical Shift

 – scale or abscissa scale

Chemical Shift Shielding  CH 3 Br < CH 2 Br 2 < CH 3 Br < TMS 90 MHz spectrum

Abscissa Scale

Chemical Shift  is dimensionless expressed as the relative shift in parts per million ( ppm ).  is independent of the magnetic field  of proton0 ~ 13 ppm  of carbon-130 ~ 220 ppm  of F-190 ~ 800 ppm  of P-310 ~ 300 ppm

Chemical Shift Charge density Neighboring group Anisotropy Ring current Electric field effect Intermolecular interaction (H-bonding & solvent)

Chemical Shift – anisotropy of neighboring group Differential shielding of H A and H B in the dipolar field of a magnetically anisotropic neighboring group  susceptibility r distance to the dipole’s center

 ~2.88  ~9-10 Chemical Shift – anisotropy of neighboring group

Electronegative groups are "deshielding" and tend to move NMR signals from neighboring protons further "downfield" (to higher ppm values). Protons on oxygen or nitrogen have highly variable chemical shifts which are sensitive to concentration, solvent, temperature, etc. The -system of alkenes, aromatic compounds and carbonyls strongly deshield attached protons and move them "downfield" to higher ppm values.

Electronegative groups are "deshielding" and tend to move NMR signals from attached carbons further "downfield" (to higher ppm values). The -system of alkenes, aromatic compounds and carbonyls strongly deshield C nuclei and move them "downfield" to higher ppm values. Carbonyl carbons are strongly deshielded and occur at very high ppm values. Within this group, carboxylic acids and esters tend to have the smaller values, while ketones and aldehydes have values 200.

Ring Current The ring current is induced form the delocalized  electron in a magnetic field and generates an additional magnetic field. In the center of the arene ring this induced field in in the opposite direction t the external magnetic field.

Ring Current -- example

Spin-spin coupling

AX system

AX 2 system

Spin-spin coupling

AX 3 system

Multiplicity Rule Multiplicity M (number of lines in a multiplet) M = 2n I +1 n equivalent neighbor nuclei I spin number For I= ½ M = n + 1

AX 4 I=1; n=3 ExampleAX 4 system

Order of Spectrum Zero order spectrum only singlet First order spectrum  >> J Higher order spectrum  ~ J

AMX system

Spin-spin coupling Hybridization of the atoms Bond angles and torsional angles Bond lengths Neighboring  -bond Effects of neighboring electron lone-pairs Substituent effect

J H-H and Chemical Structure Geminal couplings 2 J (usually <0) H-C-H bond angle hybridization of the carbon atom substituents

Geminal couplings 2 J bond angle

Geminal couplings 2 J Substituent Effects Effect of Neighboring  -electrons

Vicinal couplings 3 J H-H Torsional or dihedral angles Substituents HC-CH distance H-C-C bond angle

Vicinal couplings 3 J H-H dihedral angles Karplus curves

Chemical Shift of amino acid wn.edu/nmr/interaction/chems hf.htm

Automated Protein Chemical Shift Prediction BMRB NMR-STAR Atom Table Generator for Amino Acid Chemical Shift Assignments Chemical Shift Prediction

Example 1