The 3D HCCH-TOCSY experiment is specifically designed to correlate side-chain aliphatic proton and ¹³C resonances via ¹J(CH) and ¹J(CC) coupling constants. The experiment provides nearly complete assignments of all aliphatic ¹H and ¹³C resonances, with the exception of some resonances of the long aliphatic side chains (as Lys or Arg) for which substantial overlap remains.
 

Implementation on AVANCE spectrometers is feasible. Improved versions using pulsed field gradients (PFGs) are also available and, therefore, in such cases gradient technology is required. The experiment is applied on ¹³C-labeled proteins. Since NH protons are not involved, this experiment is usually recorded in D₂O.

The original 3D HCCH-TOCSY pulse sequence ( 90JMR425-88 ) consisted of the following steps:

1. After the initial 90º ¹H pulse, ¹H chemical shift evolution during the variable t₁ period takes place.
2. Fixed evolution delay to achieve antiphase ¹H magnetization with respect to ¹³C via ¹J(CH).
3. Magnetization transfer to ¹³C by applying simultaneous 90º ¹H and ¹³C pulses.
4. ¹³C chemical shift evolution during the variable t₂ period.
5. An isotropic mixing ¹³C period transfers magnetization along the ¹³C side chain via ¹J(CC).
6. ¹³C magnetization is transferred back to the protons by reversing the transfer steps described above.
7. Proton acquisition under ¹³C decoupling.

Several improved versions have been proposed incorporating the following modifications:

• Use of pulsed-field gradients and PEP methodology ( 93JMRB333-101 and 94JACS2203 ).
• Use of an 90º ¹³C pulse instead of the isotropic ¹³C mixing period (see 3D HCCH-COSY experiment).
• Use of heteronuclear cross-polarization ( 93JMRB333-101 and 94JACS2203 ).
• Use of homonuclear ¹³CB decoupling during the ¹³C evolution t₂ period ( 96JMRB190-113 ).
• Constant-time 3D (H)CCH-TOCSY and H(C)CH-TOCSY experiments ( 98JB89 ). Analogs 3D (H)CCH₃-TOCSY and H(C)CH₃-TOCSY experiments have been specifically designed for side-chain resonance assignment of methyl-containing residues ( 00JMR288-142 and 04JACS3710 ). Other methyl-specific MQ-(H)CCH-TOCSY and MQ-H(C)CH-TOCSY approaches described in 04JB423-30 .
• Editing and Suppression of diagonal peaks in such experiments has been also reported ( 01JB69-19 ).
• Analogs 2D HCCH-TOCSY ( 90JACS886 , 92JACS9202 and 94JACS2205 ) and 4D HCCH-TOCSY experiment ( 92JB655 ) have been also proposed.
• HCC-TOCSY-CCH E.COSY experiments (or HCCH-TOCSY-E.COSY) experiments to measure J(HH) coupling constants ( 95JACS7251 , 96JACS4388 , 96JMRB160-112 )
• 3D alpha/beta selective HC(C)H-TOCSY scheme to measure ⁿJ_CHCH coupling constants in RNAs ( 01JB117-21 )
• A forward-directed quantitative HCCH-TOCSY experiment has been proposed for the measurement of the sugar conformation in RNA oligonucleotides from CH-CH dipole-dipole cross-correlated correlation ( 99JB241 and 99JACS1956 ).
• Z-filtered HCCH-TOCSY using adiabatic TOCSY ( 00JB199-18 ).
• Carbon-detected HCCH-TOCSY pulse sequence verified in a cryoprobe ( 01CBC247 )
• 3D Doubly sensitivity-enhanced DE-MQ-(H)CCH-TOCSY experiment for the assignment of methyl resonances ( 04JB275-30 )

The 3D HCCH-TOCSY experiment can be recorded in automation mode. More details on practical implementation of the 3D HCCH-TOCSY experiment on AVANCE spectrometers can be found in the corresponding Tutorial 3D HCCH-TOCSY experiment

The HCCH-TOCSY experiment affords a 3D spectrum in which ¹H, ¹³C and ¹H chemical shifts are displayed in three independent dimensions. Cross-peaks are due to ¹H-¹³C-(¹³C)_n-¹H spins systems.