Correlation spectroscopy, COSY which method

More generally, note that the application of almost any multiple pulse sequence, where at least two pulses are separated by a time comparable to the reciprocal of the coupling constants present, will lead to exchanges of intensity between multiplets. These exchanges are the physical method by which coupled spins are correlated in 2D NMR methods such as correlation spectroscopy (COSY) [21]. 

A measurement method that is often used to determine the C-labeling of metabolites is 2D [ C. H] correlation nuclear magnetic resonance spectroscopy (COSY). This method yields relative intensities of multiplets in the NMR spectra that correspond to the relative amounts of groups of isotopomers in which the observed atom is C-labeled and the adjacent carbon atoms in the carbon backbone are either C-labeled or not (Szyperski, 1995). If the compound of which the labeling pattern is measured was synthesized by a microorganism growing on a mixture of uniformly C-labeled and naturally labeled carbon substrate, then the relative intensities of NMR fine structures can be calculated from the fractions of molecules that stem from one or more substrate molecule(s) in the feed medium. This is done using so-called probability equations (Szyperski, 1995) that require as input the fraction of uniformly C-labeled medium substrate and the fraction of naturally C-labeled carbon. 

The total correlation spectroscopy (TOCSY) techniques, which come in both 1- and 2-D versions, offer an alternative to 1-D spin decoupling and COSY methods for establishing through-bond connectivities. The important difference between the two is that TOCSY methods allow easy identification of isolated spin systems. For example, using our trusty morpholine compound once more, you can see that it is possible to identify the -CH2-CH2- spin system between the nitrogen and the oxygen atoms, these hetero-atoms, effectively isolating the protons from all others in the molecule. 

Prior to the advent of 2D methods, selective spin decoupling was used extensively in both proton NMR and in heteronuclear (especially 13C) NMR to ascertain which sets of nuclei contribute to observed spin coupling. Such information is critical to assignment of resonances and to the elucidation of the structure of an unknown molecule. 2D methods now largely supply this information much more efficiently, by correlations that depend on the existence of spin coupling. The homonuclear version of one such experiment is called COSY (correlation spectroscopy), and the heteronuclear version is known by several acronyms, most commonly HETCOR (lieferonuclear correlation). 

The value of COSY stems from its dependence on the presence of spin coupling between the nuclei involved in the correlation. As we have seen, such coupling for protons is usually limited to three or four chemical bonds, hence provides some specificity that is helpful for structure elucidation. On the other hand, useful complementary information can be obtained from longer range interactions among a set of coupled nuclei. The standard method for obtaining such information is described by two acronyms—TOCSY (for total correlated spectroscopy, which best describes the aim of the experiment) and HOHAHA (for fiomonu-clear Hartmann-Hahn, which better describes the mechanisms employed). 

TOCSY (Total Correlation Spectroscopy) is another important homonuclear 2D correlation experiment where correlations arise due to the presence of homonuclear scalar coupling.In the standard COSY experiment, crosspeaks appear for spins in which the scalar coupling occurs over typically two to four bonds. In the TOCSY experiment crosspeaks can appear for spins separated by many more bonds as long as they are part of a contiguous network of coupled spins. The correlations are effected by the application of a series of low-power rf pulses termed the spin-lock. The duration of the spin-lock period determines the extent to which the correlations are propagated through the spin system. The TOCSY experiment is a useful complement to the COSY methods for the elucidation of complex structures. 

Two-dimensional NOESY allows detection of spatial proximity between protons that are separated by less than 4.5 A. By the correlated spectroscopy method (COSY) scalar couplings between protons, which are separated by at most three (in some exceptions four) chemical bonds, are used. In the case of ribooligonucleotides such couplings consequently can only be observed between the protons of the ribose rings and the C5 and C6 protons of the pyrimidine bases of a given nucleotide. The NOESY method, however, permits the detection of contacts between the protons of different nucleotides. 

If the retention times of the analytes are known, or there is an efficient method for their detection on-line, such as UV, MS or radioactivity, stop-flow HPLC-NMR becomes a viable option. In the stop-flow technique, all the usual techniques available for high-resolution NMR spectroscopy can be used. In particular, these include valuable techniques for structure determination such as 2-dimensional NMR experiments which provide correlation between NMR resonances based on mutual spin-spin coupling such as the well-known COSY or TOCSY techniques. In practice, it is possible to acquire NMR data on a number of peaks in a chromatogram by using a series of stops during elution without on-column diffusion causing an unacceptable loss of chromatographic resolution. 

It was stated in the opening remarks of this chapter that COSY was the first two-dimensional sequence proposed, and it is in fact that given in Fig. 5.2 above, utilising just two 90 pulses. The sequence, which correlates the chemical shifts of spins that share a mutual J-coupling, is most often applied in proton spectroscopy although is equally applicable to any high-abundance nuclide. It is without doubt the mostly widely used of all two-dimensional methods, and is thus considered first. This section provides an introduction to... 

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