The 3D HCCH-COSY experiment is specifically designed to correlate side-chain aliphatic proton resonances with their attached 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 performed in D2O.

The original HCCH-COSY pulse sequence ( 90JACS888 ) consisted of the following steps:

1. After the initial 90º ¹H pulse, ¹H chemical shift evolution during the variable evolution 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 evolution t₂ period. followed by a fixed period to achieve antiphase ¹³C magnetization with respect to its ¹³C neighbors via 1J(CC).
5. A 90º ¹³C pulse transfers magnetization to its coupling partner.
6. ¹³C magnetization is transferred back to the protons by reversing the transfer steps described in points 4 and 2, respectively.
7. Proton acquisition under ¹³C decoupling.

Several improved versions have been proposed incorporating the following modifications:

• An improved version reduces indesirable artifacts and noise-like features by using selective 180 pulses and a modified phase-cycle ( 90JMR620-87 ).
• Incorporation of a constant-time ¹³C evolution period ( 91JB299 ).
• HCCH-E.COSY experiments to measure J(HH) and J(CC) coupling constants.
• Use of pulsed-field gradients and PEP methodology ( 94JACS2203 and 98JMR185-135 ). A related 2D experiment has been proposed to trace out heteronuclear connectivites at natural abundance (see 2D ADEQUATE experiment)
• Specific 2D and 3D HCCH-COSY experiments have been reported to assign deoxyribose spin systems in doubly labeled DNA ( 98JB25 ).
• A 3D H(C)CH-COSY pulse sequence with two carbon-dimensions has been recently reported ( 98JMR185-135 ).
• A modified version has been used to measure J(CP) coupling constants in RNA ( 98JMR236-133 ).
• Use of an isotropic ¹³C mixing period instead of the 90º ¹³C pulse (see 3D HCCH-TOCSY experiment).
• Use of the TROSY approach ( 98JACS6394 , 99JMR447-139 ) to be applied on aromatic ¹H-¹³C spin systems. Editing and Suppression of diagonal peaks in such experiments has been also reported ( 00JMR171-144 and 01JB69-19 ).
• TROSY-relayed HCCH-COSYfor correlation H2/H8 resonances in 13C-labeled RNA ( 01JB173-20 )
• A forward-directed quantitative HCCH experiment has been proposed for the measurement of the sugar conformation in RNA oligonucleotides from CH-CH dipole-dipole cross-correlated correlation ( 99JACS1956 ).
• A HCC-TOCSY-CCH-E.COSY experiment to measure J(HH) for ribose protons in uniformly 13C-labeled RNA ( 95JACS7251 ).

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

The HCCH-COSY 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)-¹H spins systems.

Analogs 2D HCCH-COSY experiments are also possible, yielding a COSY-type spectra. However, for larger proteins such 2D spectra show very severe overlap.