Two-dimensional Nmr Spectrum

The indirect-evolution period gives the experiment its second frequency axis. During this interval, magnetization encodes frequency information before a later coherence-transfer step and signal detection. Fourier transformation converts the time-domain data into frequency coordinates, producing a map in which resonance positions and transferred signals can be examined together. This encoding expands interpretation beyond a one-dimensional trace.

Diagonal peaks generally mark resonances associated with the same frequency relationship, whereas cross-peaks report magnetization transfer between nuclei. The transfer can reflect scalar coupling or spatial proximity, depending on the pulse sequence. Consequently, a cross-peak is not interpreted solely by its location; its meaning depends on whether the experiment is COSY, TOCSY, or NOESY and on the relationship that sequence emphasizes.

COSY and TOCSY emphasize relationships among coupled nuclei, whereas NOESY emphasizes relationships associated with spatial proximity. The choice therefore depends on whether the experiment should follow coupling-based connections or proximity-based connections. Using different spectra can provide complementary evidence for assigning resonances and interpreting biomolecular structure.

Signals that overlap along one frequency axis can be separated using their positions across two axes. The resulting pattern also links resonances through cross-peaks, allowing researchers to examine relationships rather than treating each peak as an isolated measurement. This capability is especially valuable when assigning signals in biomolecules with many resonances.

A basic workflow begins by selecting a pulse sequence suited to the relationship being investigated, then encoding magnetization during the indirect-evolution period. The sequence transfers coherence between coupled or spatially proximate nuclei, after which the measured signal is Fourier transformed along the relevant dimensions. Researchers inspect diagonal peaks and cross-peaks to interpret resonance relationships.

Researchers compare peak positions and cross-peak relationships to assign resonances within proteins or nucleic acids. COSY and TOCSY can support analysis of connected resonance patterns, while NOESY can contribute proximity-based information. These assignments provide a foundation for determining biomolecular structure and tracking how resonances change when conformation or binding state changes.

In biological research, these spectra help characterize biomolecules in solution, where resonance patterns report on molecular environments and relationships between nuclei. They also support structure determination, analysis of molecular interactions, evaluation of conformational or binding changes, and investigation of molecular dynamics. The measurements therefore help compare biomolecular states and examine how their behavior changes.