Understanding The Hsqc Pulse Sequence

Now that we know most of the basic building blocks of NMR pulse sequences, we should be able to use the coherence flow diagram (Fig. 11.9) to design an HSQC pulse sequence. It needs to accomplish the following steps:

1a. Create 1H magnetization in the X-y' plane (preparation).

1b. Transfer this magnetization from 1H to 13C via the one-bond JCH (preparation).

2. Let the 13C magnetization rotate in the X-y' plane for a period t1, allowing us to indirectly measure the 13C chemical shift (evolution).

3. Transfer the 13C magnetization back to 1H magnetization (mixing).

4. Observe the 1H magnetization directly (t2) so that the 1H chemical shift can be determined (detection).

Note that we do not start directly with 13 C magnetization because we want to take advantage of the larger (by a factor of 4) equilibrium population difference of 1H compared to 13 C, as well as the shorter T1 (faster relaxation) of1H, which will permit shorter relaxation delays. We now know a lot of tricks, and the main one we need here is the heteronuclear INEPT transfer:

1b. INEPT transfer: A delay of 1/(27) to generate an antiphase 1H doublet, followed by simultaneous 90o pulses on both the 1H and 13C channels (e.g., 500 and 125 MHz, respectively).

2. Evolution period: simply insert a delay of t1 s and repeat the experiment many times with increasingly larger t1 delays. Increment t1 each time by At1 = 1/(2 x sw1), where sw1 is the spectral width in the 13C dimension in hertz.

3. Mixing: INEPT transfer ("back" transfer). Assuming that 13C magnetization is still antiphase with respect to the directly bound proton, simultaneous 90o pulses on both the 1H and 13C channels will convert the antiphase 13C coherence back into antiphase 1H coherence. This signal differs from that at the end of the first 1/(27) delay (step 1b) in that its intensity has been modulated by the chemical-shift evolution that occurred during step 2. In other words, the 13C chemical shift has been encoded within the 1H signal.

4. Acquisition: simply turn on the analog-to-digital converter and record a 1H FID. Fourier transformation of this signal will give an antiphase doublet whose amplitude is modulated by a factor cos(^c t1).

This pulse sequence is diagrammed in Figure 11.36. Below the pulse sequence are shown the spectra that would be obtained if an FID were acquired at each stage of the pulse sequence, with 1H spectra above and 13C spectra below. The antiphase 13C signal is shown with a phase shift of 180o, for example, resulting from evolution of the 13 C chemical shift during t1.

Now that we have the basic concept of the pulse program written down, we can start to customize and enhance it, and to consider details of making it work correctly. First of

all, we have to consider the fact that the evolution periods 1/(27) and t1 serve for very different purposes: during the 1/(27) delay we want the 1H doublet to evolve from inphase to antiphase under the influence of the one-bond {1H-13C} J coupling. During the t1 period, however, we only want to see rotation of the 13 C magnetization vector in the X-/ plane under the influence of the 13C chemical shift. In other words, we want J-coupling evolution to occur during the first delay and chemical-shift evolution during the second delay. As the pulse program is designed so far, both kinds of evolution will occur during both delays, leading to lots of complications. The way to prevent certain kinds of evolution while allowing others to occur is to make each delay period into a spin echo by dividing the delay time into two equal parts separated by a 180° pulse. How we apply the 180° pulse (i.e., on which nucleus or nuclei) will determine which type of evolution is allowed and which type is refocused.

We spent a lot of time in Chapter 6 (Section 6.10) using the vector diagrams to understand the effect of 180° pulses in the center of a spin echo. This is easy to understand now that we have the product operator tools. In general, consider the effect of a 180° pulse on the in-phase and antiphase 1H and 13C operators:

Ix ^

-Ix

1H 180° y:

refocus

1H chemical-shift evolution.

Sx ^

-Sx

13C 180° y:

refocus

13C chemical-shift evolution.

2IXSZ ^

-2Ix Sz

1H 180° y or

13C 180

D: refocus J-coupling evolution.

2IxSz ^

2Ix Sz

1H 180°y and 13C 180°:

no effect on J-coupling evolution.

Reversing the sign of these operators in the center of a spin echo leads to refocusing of the evolution. This is an easy way to remember how to design a heteronuclear spin echo: use a 1H 180° pulse alone to refocus all but 13C chemical shift evolution; use a 13C 180° pulse alone to refocus all but 1H chemical-shift evolution; use simultaneous 1H and 13C 180° pulses to refocus all but JCH evolution. You can go through the full product operator analysis of each kind of spin echo, and you will find that the sign changes shown above are the crucial differences that control what refocuses and what continues to evolve in the second half of the spin echo.

For the first delay of 1/(2J) in Figure 11.36, we insert simultaneous 180° pulses in the center on both 1H and 13C (Fig. 11.37). The 1H chemical-shift evolution "sees" the 1H 180° pulse in the middle and reverses direction (vH in the first half, — vH in the second half). The J coupling evolution "sees" the simultaneous 180° pulses on both channels and their effects cancel out (2Iy Sz ^ 2[—Iy ] [—Sz ]) so the J-coupling evolution continues unabated in the second half (J in the first half, J in the second half). We used the same strategy in the INEPT experiment (Chapter 7). At the end of the 1/2J period we have antiphase 1H coherence, which is transferred to antiphase 13 C coherence by the simultaneous 90° pulses on 1H and 13C (2IySz ^ —2SyIz).

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