The 3D HCACO experiment  is specifically designed to correlate intraresidue ¹HA, ¹³CA and ¹³CO chemical shifts by means of the ¹J(CH), ¹J(CA,CO) coupling constants.

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 ¹³C-labeled proteins. Since NH protons are not involved, this experiment is performed in D₂O.

The original 3D HCACO pulse sequence ( 90JMR496-89 ) consisted of the following steps:

1. Initial transfer from ¹H to ¹³C via ¹J(CH) using an INEPT pulse sequence.
2. ¹³C chemical shift evolution during a variable t₁ period followed by a period to achieve antiphase ¹³CA magnetization with respect to ¹³CO spins.
3. Magnetization transfer from ¹³CA to ¹³CO applying two simultaneously 90º ¹³CA and ¹³CO pulses.
4. ¹³CO chemical shift evolution during the variable t₂ period in a HSQC-like way.
5. Magnetization is transferred back to the HA protons by reversing the steps 2 and 1, respectively.
6. Proton acquisition under ¹³CA decoupling.

Several improved versions have been proposed incorporating the following modifications:

• Variable constant-time CA periods have been proposed in order to remove the antiphase doublet character from the CA lineshape ( 91JMR209-94 ). Other constant-time schemes have also been reported ( 95JMRB189-107 ).
• Improved sensitivity ( 92JB103 ).
• Modified version to suppress undesired artifacts ( 93JMRB103-102 ).
• Modified versions to achieve further ¹H connectivities via ¹³C-¹³C COSY and TOCSY mixing blocks ( 92JMR653-99 , 95JMRB80-109 , and 96JB247 ) (3D HCACO-TOCSY experiment).
• Use of pulsed field gradients ( 95JMRB285-108 , 96JB247 , and 97JMR270-126 ).
• Carbon-detected HCACO pulse sequence verified in a cryoprobe ( 00JACS3554 , 01CBC247 ). Related multiple-quantum CO-detected HACACO experiment for large proteins ( 03JB147-25 and 03JB69-26 )
• A similar experiment has been proposed in which the transfer mechanism has been reversed (3D COCAH experiment) ( 94JMRA102-107 ).
• HMQC version of the HCACO experiment which offer improved relaxation behavior using the multiple-quantum narrowing approach ( 00JMR407-143 )

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

The HCACO experiment affords a 3D spectrum in which ¹HA, ¹³CA and ¹³CO chemical shifts are displayed in three independent dimensions. Intra-residue correlations are due to the ¹J(CH) +  ¹J(CA,CO) mechanism.

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