Evolution delay

Dummy scans are the preparatory scans with the complete time course of the experiment (pulses, evolution, delays, acquisition time). A certain number of these dummy scans are generally acquired before each FID in order to attain a stable steady state. Though time-consuming, they are extremely useful for suppressing artifact peaks. 

Figure 22 Pulse sequence of the HMBC-RELAY experiment. Filled and open bars represent 90° and 180° pulses, respectively. All other phases are set as x, excepted otherwise stated. A two-phase cycle x, —x is used for the pulse phases (j>, and

A number of 2D NMR experiments have been developed which allow the study of slow exchange phenomena. The most common (Exchange Spectroscopy, EXSY), is based on the standard pulse sequence 90°x-fi-90°x-tm-90°x - FID(t2), where is an evolution delay, is the mixing time, and tj is the detection period [175, 176]. 

The second (sine) term produced by the evolution delay has all the information we need—it is antiphase 13C coherence labeled with the 13C chemical shift in t —but it is lost because its phase (S ) causes it to be unaffected by the 90° 13C pulse at the end of t. Effectively we are throwing away half of our signal at this point. A new method was developed to save this wasted signal and boost the sensitivity of HSQC and many other experiments. These modified pulse sequences are called sensitivity enhanced or sensitivity improved (Bruker adds si to the pulse sequence name) and the strategy is called preservation of equivalent pathways (PEP) because the two terms are equivalent except for their phase. 

To make this into a 3D experiment we need to create a third time domain, in this case a time domain that encodes the chemical shift of the Hn proton. We simply stop for a moment on our journey from 15N SQC to Hn SQC to Ha and side-chain H coherence, at the point where we have an Hn coherence, and insert an evolution delay to indirectly record the chemical shift of the Hn- The pulse sequence is shown in Figure 12.46 (center) and the coherence pathway is diagramed in Figure 12.47. The new evolution delay is called f2 because it is the second independent time domain, forcing us to rename the direct time domain of the FID as 3. In the center of the t2 evolution delay there is a 15N 180° pulse to reverse the 1Jnh coupling evolution so that the Hn will not be split by 15N in the F2 dimension, just as the t evolution delay includes a 180° pulse in the center to decouple ... 

The 3D Fourier transform is performed in three steps, starting with the directly detected time domain 13. For example, for a 3D TOCSY-HSQC we might have 100 t values (200 FIDs 100 real and 100 imaginary) and 32 values (64 FIDs) for a total of 12,800 FIDs. The t dimension is H and the t2 dimension is 15N. The acquisition order is the order in which the evolution delays are incremented in this example the first delay (t, H evolution) is incremented first and constitutes the inner loop. That means that we go through all 1001 values first, keeping the t2 delay fixed at the first value (usually zero). Then we repeat the whole process with the t2 delay set to the second value, and so forth. 

The resolution in the indirectly detected heteronuclear dimension (F ) depends on the number of increments of the evolution delay, i.e. the number of free... 

Fig. 2. Pulse sequence for the HMBC-RELAY experiment proposed by Sprang and Bigler to differentiate V from "i (n = 3, 4) long-range heteronuclear correlations. Ibises are represented as solid (90°) or open (180°) bars. All pulses are x except for the y pulse in the composite pulse and the last 90° proton pulse. A two step phase cycle, x, —x is used for the pulse phases i, 2, and the receiver phase. Two FIDs are recorded for each increment of the evolution time, ti with the phase i set as X, —X and —x, x, respectively (interleaved detection) with the data stored separately. Delays are set as D4=1/[4Vch]. D6 = 1/[4 /hh]> and D5 = y2["AH]- The evolution delay, DO, must remain equal for both evolution periods. The gradient ratio optimized for is 4, 4,...

The heteronuclear polarization transfer step The simplest sequence unit to create coherence transfer from a sensitive nucleus I to a insensitive nucleus S is a 90°(I) pulse and a 90°(S) pulse enclosing a scalar coupling evolution delay. This unit forms part of many pulse sequences including the HMQC and the HMBC experiment. 

OGradients and coherence evolution delays are not specific to 2D experiments but the correct setup of these parameters is particularly important for 2D experiment. 

The PENDANT sequence is an extended version of the INEPT sequence for the detection of quaternary carbon atoms in a similar way that the DEPTQ sequence is an extension of the standard DEPT sequences. Initially the only difference between PENDANT and the refocused INEPT sequence is an extra 90° l C pulse executed simultaneously with the first 90° H pulse. Upon closer inspection a second difference is apparent, the refocusing period delay d3 in the INEPT sequence is 3/(8 H(C, H)) and 5/(8 U(C, H)) for PENDANT. Compared to the DEPT sequence, the modifications necessary to detect quaternary carbon atoms are easier to implement in the refocused INEPT sequence. The refocused INEPT sequence has two periods for refocusing chemical shift evolution so that the quaternary carbons atoms coherences which are generated by the initial 90° pulse evolve under the chemical shift evolution during the free precession periods and are refocused immediately before the acquisition period. In contrast the DEPTQ sequence requires an additional coherence evolution delay... 

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