112 INSTRUMENTAL METHODS
Following this, each FID is Fourier - transformed to produce perturbed spectra.
These are stacked, and the data sets at each frequency point are transformed
once again. The results give data that are displayed on two frequency axes,
with the peaks represented by contours on a relief map. The two - dimensional
plot contains connectivities between the different nuclei. The connectivities
or correlations may involve spin – spin coupling of nuclei through covalent
bonds or show spatial proximity through nuclear Overhauser (NOE) effects.
A simple conceptual diagram of the 2D experiment is shown in Figure 3.15.
3.4.9 Two - Dimensional Correlation Spectroscopy (COSY) and Total
Correlation Spectroscopy (TOCSY)
Three two - dimensional NMR methods have been commonly used in deter-
mining the three - dimensional structure of proteins, although there are
many variations that we will not discuss here. The website at http://www.
genomicglossaries.com/content/nmr_email.asp contains a glossary and defi ni-
tions of some other NMR methods. Two methods, COSY and TOCSY, transfer
magnetization through the chemical bonds between adjacent protons. These
techniques are discussed in this section. The third method, nuclear Overhauser
effect spectroscopy (NOESY), is discussed in Section 3.4.10. COSY or correla-
tion spectroscopy is a technique with many variations, all of them depending
upon the existence of spin – spin coupling between nuclei. The coupling pro-
vides responses relating to the chemical shift positions of coupled nuclei. Its
equivalent in one - dimensional spectroscopy would be a series of double
resonance experiments at each multiplet in the spectrum. One can perform
homonuclear (probing couplings between the same nuclei, i.e.,^1 H –^1 H)
or heteronuclear (probing connectivities between different nuclei, i.e.,
(^1) H – (^13) C) COSY experiments.
In the pulse sequence for a homonuclear COSY experiment, the fi rst 90 °
pulse fl ips the z magnetization into the x direction and into the xy plane. Con-
sidering an AX spin system (one in which the nuclei have very different chemi-
cal shifts) having two doublets due to spin – spin coupling, the magnetization
will include four components precessing at different frequencies. During a
Figure 3.15 Conceptual diagram of a two - dimensional (2D) NMR experiment.
(Adapted with permission of Nelson Thornes Ltd. from Figure 8.1 of reference 21 .)
pre-experimental
delay, D
preparation mixing
t 1
evolution
t 2
FID
detection