The 2D TOCSY (TOtal Correlation SpectroscopY)  experiment (also called as HOHAHA (HOmonuclear HArtmann-HAhn) experiment) permits to assign a whole spin system regardless of the exact topology. In comparison with RELAY experiments, TOCSY experiment requires only a single mixing period and less detailed knowledge of spin system topology and couplings. In addition, it generates phase-sensitive spectra without intensity alternation, giving better sensitivity for poorly-resolved multiplets. TOCSY experiment can be used to obtain subspectrum characteristic of part of a molecule that has no scalar couplings to other spins in the molecules (for instance, to sequence polysaccharides, peptides or proteins).

Easy implementation on any AVANCE spectrometer.

The basic pulse sequence of the TOCSY experiment ( 85JACS2820 ) follows the same principles of the homonuclear 2D experiments. After a excitation 90º 1H pulse, trasnverse magnetization evolves during a free variable evolution t1 period. Instead of relaying magnetization via antiphase states, the TOCSY experiment uses an isotropic mixing sequence to transfer magnetization between spins via the strong scalar coupling Hamiltonian, with the result that in-phase magnetization can be transferred through several couplings during the mixing time (see TOCSY block) . Proton detection is performed as usual. Coherence transfer in TOCSY experiments proceeds from the in-phase magnetization and, therefore, the commonly antiphase cancellation in COSY-type experiments are not a problem here. The isotropic mixing is usually performed applying a WALTZ, MLEV or DIPSI pulse train and it can be inserted into two z-filters and, in such case, isotropic mixing is performed on the longitudinal magnetization  ( 87JMR557-74 and 88JMR358-76 ). The TOCSY pulse sequence can also be combined with some solvent suppression scheme: PEP methodology can also be incorporated in TOCSY experiments to improve the sensitivity  ( 90JMR72-88 and 93AR1 ).

It is possible to determine the number of successive couplings through which magnetization giving rise to a particular cross peak in a TOCSY spectrum has been transferred (Taylored TOCSY). In its basic implementation ( 95JMRA188-114 ), it is based on recording a series of TOCSY spectra (between 10 or 20 spectra) in which the mixing time is steadily increased. The intensity of a particular cross-peak follows a specified transfer function that, from a fitting procedure, indicates how many successive couplings are involved. Using the accordion spectroscopy, it has been shown that this information can be obtained from a single experiment ( 95JMRA35-115 ). In the accordion spectrum the mixing time is incremented in concert with the evolution time. Thus, the nature of cross-peak in the F1 dimension reflects the evolution during both t1 ans the mixing time. This alters the lineshape of cross-peak and its analysis permits to obtain the specified transfer function.

Pulsed field gradients can be incorporated in the ge-2D TOCSY experiment.

2D TOCSY experiments have been also applied to 11B ( 96POL359 and 95POL961 ).

The 2D TOCSY can be recorded in routine/automation modes. The most important parameter to consider is the length of the mixing time. By carrying out a series of experiments with successively longer mixing times (for instance, from 20 to 120 ms), it is possible to trace out connectivity pathways through the whole spin system. Some practical details can be found in 89MRC529 .More details on practical implementation of 2D TOCSY experiments on AVANCE spectrometers can be found in

Tutorials: 2D homonuclear experiments

Tutorials: 2D gradient-based homonuclear experiments

Tutorials: 2D gradient-based homonuclear experiments in H₂O

TOCSY experiment affords a 2D chemical shift correlation map similar as in a RELAY-type experiment in which both direct and relayed connectivites are present.

A problem with the TOCSY experiment of large biomolecules can be the presence of cross-relaxation (ROESY effects). However, they can be easily differenciate because TOCSY cross-peaks are positive and ROESY cross-peaks are negative with respect to the positive diagonal peaks. Designed spin-lock sequences has been designed to circumvent this problem (the so-called CLEAN-TOCSY experiment) ( 88JACS7870 and 92JMR184-99 ).

Relayed coherences can also be established using the 2D RELAY experiment.

Related experiments:

2D homonuclear experiments

2D gradient-based homonuclear experiments

Some interesting articles about the principles of the TOCSY experiment: 96CONC229 and 94CONC115 .