The 3D HBHANH experiment is specifically designed to correlate the ¹H and ¹⁵N amide resonances of one residue with both ¹HA and ¹HB resonances of the same residue and of its preceding residue via the intervening ¹³CO,  ¹³CA and ¹³CB spins by means of the ¹J(NH), ¹J(N,CA) (and ²J(N,CA)), ¹J(CA,CO),  ¹J(CH) and optional ¹J(CA,CB) coupling constants. This experiment is an extension of the 3D CBCANH experiment and is closelr related to the 3D HBHA(CO)NH experiment

Implementation on AVANCE spectrometers equipped with a third channel. Improved versions using pulsed field gradients (PFGs) are also available and, therefore, in such cases gradient technology is required.

The experiment is applied on ¹⁵N,¹³C-labeled proteins. Because the amide (NH) protons are involved, the 3D HBHANH experiment must be recorded in H₂O.

The original 3D HBHANH pulse sequence ( 94JMRB196-105 ) consisted of the following steps:

1. After a 90º ¹H pulse, ¹H chemical shift evolution takes place during a variable t₁ period.
2. Antiphase ¹H magnetization with respect to ¹³C is also allowed to evolve and polarization transfer is achieved by applying two simultaneous 90º ¹H and ¹³C pulses. Then, After evolution of ¹³CA-¹³CB couplings, a 90º ¹³C pulse transfers magnetization from ¹³CB to ¹³CA.
3. Fixed evolution delay to achieve antiphase ¹³CA magnetization with respect to ¹³CO via ¹J(CA,CO) followed by  magnetization transfer to ¹³CO by simultaneous 90º ¹³C and ¹³CO pulses.
4. Fixed evolution delay to achieve antiphase ¹³CO magnetization with respect to ¹⁵N via ¹J(N,CO) followed by  magnetization transfer to ¹⁵N by simultaneous 90º ¹⁵N and ¹³CO pulses.
5. ¹⁵N chemical shift evolution during the constant-time t₂ period. Magnetization is finally transferred back to the NH protons by a retro-INEPT pulse train and proton acquisition is recorded under ¹⁵N decoupling.

Several versions have been proposed incorporating the following modifications:

• Use of WATERGATE:
• Similar 3D H(CCO)NH or 3D H(C)(CO)NH-TOCSY experiments use isotropic ¹³C mixing instead of a 90º COSY-type pulse to transfer magnetization from the aliphatic ¹³C to ¹³CA.
• A 4D HCC(CO)NH experiment has also been proposed ( 93JB349 ).

More details on practical implementation of the 3D  HBHANH experiment on AVANCE spectrometers can be found in the corresponding Tutorial 3D HBHANH experiment

The  HBHANH experiment affords a 3D spectrum in which ¹H, ¹⁵N and ¹HA/¹HB chemical shifts are displayed in three independent dimensions. Intra-residue and sequential connectivities are due to ¹J(NH) +  ¹J(N,CA) +  ¹J(CA,CO) + ¹J(CH) + optional ¹J(CA,CB) and ¹J(NH) +  ²J(N,CA) +  ¹J(CA,CO) + ¹J(CH) + optional ¹J(CA,CB)

See list of 3D triple-resonance NMR experiments for doubly-labeled proteins.