2D: Heteronuclear: X-detected

HETCOR
HETCOR-proton decoupling
HETCOR-Long range
COLOC
HETCOR-RELAY (using HOHAHA)

The 2D experiments presented here are shift correlation experiments involving two different nuclei interacting with scalar coupling constant JX-H. These experiments were very popular in the 80's but with the advance made in NMR, they have now been replaced by the highly sensitive reverse experiments which detects the highly sensitive protons instead of the low-gamma low-sensitivity X nuclei.

HETCOR (HETeronuclear COrrelation)

The HETCOR experiment is similar to the COSY experiment with the exception that it concerns two different nuclei. The simplest pulse sequence that can be proposed for such 2D involves simply two 90` pulse, the last one being applied simultaneously to both nuclei. The first pulse excite proton nuclei whose chemical shift is detected during the evolution period t1. The proton signal of interest is coupled not only to other protons but also to the heteronuclei (and therefore dedoubled with a large coupling constant). The last proton pulse transfers proton magnetization to the X-nuclei (through scalar coupling) whose intensity will be modulated by the proton chemical shift. Of course, this experiment is not very useful as such as we would like to detect decoupled X nuclei at least!

The "real" HETCOR pulse sequence involves refocusing of the heteronuclei coupling during the evolution time (t1) by applying a 180 ` pulse to the X-nuclei so that the protons will retain only the homonuclear coupling constant. The next 1/2J delay insures an efficient signal intensity transfer to the X-nuclei by developing the antiphase character for the heteronuclear coupling. The simultaneous 90 degree pulses on proton and on the X-nuclei transfers the magnetization to the X-nuclei (antiphase components like in INEPT sequence) whose intensity is modulated by the proton chemical shift. The last delay allow for the X-antiphased components to refocus so that the proton decoupler can be turn on for the subsequent detection period (t2). As the X-nuclei can be coupled to 1, 2 or 3 protons, an "optimum" refocusing delay that takes into account the various multiplicities for the X-nuclei (doublet, triplet or quartet with INEPT enhancement) must be setup. The refocusing delay used in standard sequence is about 1/3J (good intensity on CH, CH2 and CH3 type of carbons). This INEPT based HETCOR have been used extensively in the past but is the sensitivity of this technique much lower if we compare with the reverse experiments (HMQC and HSQC).

HETCOR with proton decoupling

If the pulse sequence is modified to include a bird pulse in the middle on the evolution time (instead of an ordinary 180` pulse on the X-nuclei), the proton-proton coupling gets also decoupled resulting in higher sensitivity. In the BIRD sequence, the first proton 90` pulse stores the proton magnetization along the Z-axis. The net result of the BIRD sequence is that it inverts the labels of protons bound to C-12 while keeping intact the labels of the protons bound to C-13. This selective inversion will produce homonuclear decoupling at the end of the evolution period. The 180` pulse (applied to both nuclei) inverts the protons as well as the heteronuclear labels insuring also the refocus of the heteronuclei coupling. The last 90` pulse (of BIRD element) restores the proton magnetization which continue to evolve for the second half of the evolution time. The proton magnetization is then transferred back using the usual INEPT transfer.

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These HETCOR and XCOR sequences can be further improved by including refocusing pulses in the INEPT transfer periods.

COLOC (COrrelated LOng range Coupling)

Modification of the delays in HETCOR can provide long range coupling information (coupling between proton and carbon through 2-3 bonds). The problem with these smaller heteronuclear couplings is that the pulse sequence gets dramatically longer, reducing the sensitivity of the experiment. An improvement to the HETCOR sequence is to make the evolution time constant and to include in it the INEPT polarization period (the net result is that the 180` pulse pair moves as a function of the evolution time such that at the end of the delay the proton magnetization coupled to the X-nuclei have acquired an antiphase character. Then, the INEPT polarization (double 90` pulse applied to both nuclei) can take place without further delay. This modification makes the pulse sequence shorter and more sensitive (less relaxation during the pulse sequence).

This sequence is quite insensitive and have been replaced by the inverse experiment: HMBC.

HETCOR-RELAY

This experiment combines the power of HETCOR with Relay-COSY. Although this experiment can be done by adding a "COSY-RELAY" step to the regular HETCOR sequence, the best way to do this experiment is by using a TOCSY mixing period after the HETCOR evolution period as shown in the figure below.

This pulse sequence is as sensitive as the HETCOR but again, with the evolution of NMR, these experiments are now best done in the inverse experiment mode (HMQC-TOCSY or HSQC-TOCSY).