NMR Spectroscopy is one of the most powerful techniques for structural elucidation and solution conformational analysis of Organic (and inorganic) compounds and biomacromolecules.  NMR spectroscopy gives detailed information about molecules and their environment based on the interactions of nuclear magnetic moments with electromagnetic radiation.

Information Content of an NMR Spectrum

1.         The Chemical Shift.  This is due to a difference in the chemical (bonding) environment of the nuclei (e.g. ¹H).  The external magnetic field, B₀, causes the electrons to circulate within their atomic orbitals and this induced motion generates a small magnetic field B` in the opposite direction to B<sub>0</sub> (similar to an electric current passing through a coil of wire).  Thus nuclei are shielded from the external magnetic field by their surrounding electrons to a different extent (depending on their local electron distribution) [B = B<sub>0</sub>  - B`].  The chemical shift is commonly defined as the difference in resonance frequencies between a reference nucleus (n_ref) and the nucleus of interest (n), by a dimensionless parameter d, which is a molecular property, independent of the magnetic field used to measure it.

d = 10⁶ (n - n_ref) / n_ref

2.         Integration.  This Refers to the area under the peak, typically automatically calculated by the program, and gives information about the number of nuclei that gave rise to that peak.

3.         Splitting.  This refers to the splitting of the NMR peaks in to 2, 3, 4, 5, etc. (doublet, triplet, quartet, quintet, etc.) and gives information about the number of chemically and magnetically equivalent nuclei. The n + 1 rule states that for a nuclei of spin ½, n equivalent neighbors will split the signal of a spin ½ nuclei in to n + 1 lines.

4.         Scalar (J)-coupling.  This is a powerful feature that refers to the distance between the splitted peaks (above) in units of Hz.  By making use of the Karplus equation [1] the value of the coupling constant can be converted to geometry information (dihedral angle or torsion).

5.         Nuclear Overhauser Effect (nOe).  This is a special kind of NMR experiment that

can be done in 1D or in 2D.  It refers to the enhancement of NMR signals of the nuclei when a nuclei to which they are interacting through space (as opposed to through bonding in the case of J-coupling interactions) is irradiated with a radio frequency.  It gives information about how far apart the different nuclei are from the irradiated nuclei through the inverse r⁶ relation of the distance between the nuclei to the intensity of the nOe signal.

6.         Dipolar (D) Coupling.  This refers to the interaction between nuclear dipole moments through space.  In liquids this interaction averages out to zero because of the fast tumbling of the molecules.  But in solids, where fast tumbling of the molecules does not take place, the dipolar coupling does not average out to zero and is especially important in solid-state conformational analysis of molecules.  As a significant amount of the proteins in the cell are membrane proteins that reside in liquid crystalline membrane environment, solid-state NMR spectroscopy is rapidly becoming an important technique for conformational analysis of membrane proteins.

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Nuclear Magnetic Resonance (NMR) Spectroscopy in Organic Chemistry Laboratory

Organic chemistry students at Kenyon College routinely use our 300 MHz Bruker NMR spectrometer to characterize the compounds they synthesized and to identify an unknown compound as part of their Organic Chemistry Lab I (CHEM 233) and II (CHEM 234).  A sample experiment is discussed below.

Sodium borohydride reduction of 4-tert-butylcyclohexanone

In this experiment, students prepare 4-tert-Butylcyclohexanol by reducing 4-tert-butylcyclohexanone with a methanolic solution of sodium borohydride.  This reaction, shown below, gives a mixture two products and NMR spectroscopy is used to determine the percentage composition of the two products.  In addition, they use Karplus equation (shown below) to calculate three dihedral angles (H-C-C-H) of the two products hence gaining insight in to the conformations of these two molecules.

Scheme 1. Reduction of 4-tert-Butylcyclohexanone with sodium borohydride

Chart 1.  Structures of the two products

Karplus Equations 3JH-C-C-H = 10 cos2 q for 0 £q £ 900, and 3JH-C-C-H = 12 cos2 q for 90 £q £ 1800

Table 1.  Definition of dihedral angles, the expected dihedral angle values, and the expected values of the scalar coupling constant.

Figure 1.  ¹H NMR spectrum of the crude product of sodium borohydride reduction of 4-tert-Butylcyclohexanone.

Figure 2.  Expanded (He, Ha) ¹H NMR spectrum of the crude product of sodium borohydride reduction of 4-tert-Butylcyclohexanone.

Nuclear Magnetic Resonance (NMR) Spectroscopy in Advanced Biochemistry at Kenyon College

Advanced Biochemistry (CHEM 460) students at Kenyon College use our 300 MHz Bruker NMR spectrometer to study the solution conformations of carbohydrates and peptides.  The most detailed information you can get about the structure and dynamics of a molecule (e.g. a protein) in solution is from NMR Spectroscopy.  They perform multinuclear 1D and 2D NMR spectroscopy experiments routinely.  Sample spectra are shown below.

Figure 3.  Low energy conformation of sucrose.

Figure 4.  ¹H NMR spectrum of the disaccharide sucrose.

Figure 5.  2D Correlated NMR spectroscopy (COSY) spectrum of the disaccharide sucrose.

Figure 6.  2D Heteronuclear multiquantum correlated NMR spectroscopy (HMQC) spectrum of the disaccharide sucrose.

Figure 7.  2D ROESY spectrum of a trisaccharide.