NMR arises from the fact that certain atomic nuclei have a property called “spin”

“Spin” is caused by circulating nuclear charge and can be thought of as bar magnet that adopts a preferred orientation in the presence of a static magnetic field

Static Magnetic Field Generated by the NMR Spectrometer

Nucleus of NMRActive atom

NMR spins adopt specific quantitized states; for spin ½ nuclei, the type of nuclei most commonly studied, there are two such states, and .

corresponds to preferred low energy configuration, while corresponding to the non-preferred high energy configuration.

Nuclear Magnetic Resonance (NMR)

Energy can be applied by the NMR spectrometer in the form of an oscillating magnetic field at a precisely defined frequency (and hence energy) that induces transitions from to .

Oscillating magnetic field is applied through the probe, which basically consists of a coil surrounding the NMR tube attached to a user controlled oscillating frequency source on the spectrometer console.

Nuclear Magnetic Resonance (NMR)

NMR Chemical Shifts

= physical constant for a given type of nucleus (ratio of magnetic moment and angular momentum)

h = Planck’s constant

Bo = static magnetic field strength

Prediction, based on the fact that all nuclei in the sample are placed in the same magnet, is that all nuclei of one type (1H,

13C, 15N, 31P etc) would have exactly the same NMR frequency

Predictions Do Not Match Reality

= chemical shielding tensor

Frequency

Ile in D2O

12

1

HDO

121

(Acquisition time = 30 s)

Chemical Shielding

Shielding arises from the various ways by which electrons“shield” the nuclear spin from the external magnetic field (Bo)

Physical mechanism relates to induced circulation of electrons that oppose static magnetic field (Lentz’ Law)

Shielding (tensors) can be determined through ab initio calculations. This, however, is computationally expensive, and not routinely applied to large molecules, such as proteins.

Classic Approaches to Shielding Local electronic structure; electronegativity of attached groups, bond lengths, bond angles, and conformation (dihedral angles)

Anisotropy of local groups (circulating electrons from aromatic rings for example)

Hydrogen bonds

Electric field effects that polarize bonds

Chemical Shielding Trends for Protons

Functional Groups

Proteins

Frequency

Frequency

Chemical Shifts Can Change Dramatically with Changes in Conformation

8 M Urea

No Urea

Chemical Shielding & Chemical Shifts

Recall

Bo field dependence of frequency makes comparison of spectradifficult from one instrument to another

Hence, report relative ’s, not absolute ’s

Chemical Shift (ppm) = =

peak = frequency of signal of interestref = frequency of reference signal

IUPAC-IUB Shift Standard for Proteins

Sodium-2,2-dimethyl-2-silapentane-5-sufonate (DSS)

J-coupling

Ha

Hb

J-couplings in Ile

Ile in D2O

1

2

1

1

2 1

1

NMR Active Nuclei in Biomolecules

Sensitivity of NMR

& spin states willassume a Boltzman distribution

Implications: Highest sensitivity w/ higher & higher Bo

1D 13C Natural Abundance Spectrum of Ile

Ile in D2O (1H Decoupled)

(Acquisition time = 4 hr)

1

2 1

13C ppm13C ppm

112

CO

13C ppm