Axial peaks

Whilst there exist a whole host of artefacts that can arise in 2D spectra according to the details of the experiment, the two most likely to be encountered are briefly considered here. Both arise from instrumental imperfections, and how significant they are to you will be somewhat dependent on your instrument and its performance, but in any case it is useful to be able to recognise these artefacts if and when they appear. 

F ure 5. Bands of noise parallel to the fi axis (ti-noise) often appear in 2D spectra. 

At the most basic level one has to address three fundamental questions when setting up an experiment 1) will the defined parameters enable (or limit) the detection of the desired information and exclude the unwanted 2) how long will it take and do I have enough time and 3) how much storage space is required for the data and is this available Ideally we would wish to acquire data sets rapidly and we would like these to be as small as possible for speed of processing and ease of handling yet still able to provide the information we require, so we set up our experiment with these goals in mind. 

The key lies in deciding on what level of digitisation is required for the experiment in hand. The first point to notice is that adding data points to extend the t2 dimension leads to a relatively small increase in the overall length of 

Experiment Spectral width (ppm) N(t2) N(t.) Hz/pt (tz) Hz/pt (ti) Experiment time Raw data-set size 

The fundamental premise of 2D NMR is that magnetization precesses during the fj period and, as we have seen, thus introduces a modulation into the magnetiza- 

Digitisation of data during a 2D experiment is subject to the same thermal noise arising from the probe head and preamplifier as in a ID experiment, and this contributes to the noise baseplane observed in the 2D spectrum. There also exists a particularly objectionable artefact associated with 2D experiments, referred to as noise (note the here refers to the evolution period and should not be confused with the relaxation time constant). This appears as bands of noise parallel to the/i axis where an NMR resonance exists, and it is sometimes this that limits the observation of peaks in the spectrum rather than the true thermal noise. Indeed, it appears that the very earliest work on 2D NMR was unpublished due to excessive ti noise present in the spectra. Generally speaking, this is caused by 

Significant reductions in the level of fi noise can also be obtained in certain experiments where signal selection is achieved by pulsed field gradients, although homonuclear experiments such as COSY show little benefit in this respect, more notable gains being apparent 

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All standard pulse sequences for these experiments include some mechanism for selecting xH-X pairs and suppressing the other XH signals (phase cycling and/or pulsed field gradients, see below). As a further advantage, residual signal intensity of protons not bound to X (because of imperfect suppression) will not lead to a cross peak in the xH,X plane after Fourier transformation, but merely contribute to axial peaks at the spec-... 

Because of the presence of sleam voids in lire upper part of [he core, there is a natural characteristic for a BWR to have the axial power peak in the lower part of the core. During the early part of an operating cycle, bottom-entry control rods permit a partial reduction of this axial peaking by locating a larger fraction of the control rods in the lower part of the... 

Figure 10 shows the Fourier transformations of intrinsic heterodyne detected time domain two-dimensional Raman spectra of (a) CCU, (b) CHCI3, and (c) a 50 50 molar ratio CCL CHCE mixture. Only quadrants I and II are shown, since the other two quadrants are equal by inversion symmetry. Aside from the diagonal and axial peaks, there... 

Application of NOESY-HMQC to the nuclease sample replaces the 2D NOESY spectrum of Fig. 12.12a by a 3D spectrum that can be displayed in a cube but is more easily interpreted as a set of planes, as indicated schematically in Fig. 12.14. Two of the NOESY planes obtained in the nuclease experiment are illustrated in Fig. 12.126. Clearly, the 3D experiment is successful in editing the uninterpretable spectrum of Fig. 12.12a into manageable pieces. Moreover, each NOESY plane is labeled by the chemical shift of the 15N that is coupled to one of the protons, so additional useful information may be available if that 15N chemical shift can be related to structural features in the molecule. Note that a proton not coupled to any 15N generates an axial peak, rather than a cross peak, in the 3D spectrum, but phase cycling is used to remove axial peaks. Also, displays other... 

Figure 5.23. Axial peaks in a COSY spectrum form a band of signals along f2 at (a) the midpoint of fj (States or absolute-value data sets) or (b) at the high-frequency edge of the spectrum (TPPI data sets).

Table 5.3. The basic two-step phase cycle for the elimination of axial peaks...

Scenario (a) transplants acquisition parameters from a typical ID proton spectrum into the second dimension leading to unacceptable time requirements, whereas (b) and (c) use parameters more appropriate to 2D acquisitions. All calculations use phase cycles for f quad-detection and axial peak suppression only and, for (b) and (c), a recovery delay of Is between scans. A single zero-filling in f] was also employed for (b) and (c). 

The basic components of the INADEQUATE phase cycle comprise doublequantum filtration and fi quadrature detection. The filtration may be achieved as for the DQF-COSY experiment described previously, that is, all pulses involved in the DQ excitation (those prior to ti in this case) are stepped x, y, —X, —y with receiver inversion on each step (an equivalent scheme found in spectrometer pulse sequences is to step the ftnal 90° pulse x, y, —x, —y as the receiver steps in the opposite sense x, —y, —x, y, other possibilities also exist). This simple scheme may not be sufficient to fully suppress singlet contributions, which appear along fi = 0 as axial peaks and are distinct from genuine C-C correlations. Extension with the EXORCYCLE sequence (Section 7.2.2) on the 180° pulse together with CYCLOPS (Section 3.2.5) may improve this. Cleaner suppression could also be achieved by the use of pulsed field gradients, which for sensitivity reasons requires a gradient probe optimised for C observation. 

The scheme of Fig. 6.31b has been widely used to produce absolute-value shift correlation spectra, and is often referred to as HETCOR or hetero-COSY. Conversion to the preferred phase-sensitive equivalent (of which various forms have been investigated [55]) requires the reintroduction of the simultaneous 180°( H, C) pulses into the midpoints of both Ai and A2 to remove chemical shift evolution during these periods, exactly as in the full refocused INEPT. In addition, the incorporation of the States or TPPI phase cycling of the 90° proton pulse of the polarisation transfer step is required. Suppression of axial peaks is through the phase alternation of the final proton pulse together with the receiver... 

Term a has no evolution as a function of q and so will appear at Fl = 0 in t2 it evolves at Qs. This is therefore an axial peak at FVF2 = 0, This peak arises from z-magnetization which has recovered during the mixing time. In this initial rate limit, it is seen that the axial peak is zero for zero mixing time and then grows linearly depending on Rs and oIs. 

When the SHR method is used, axial peaks (arising from magnetization which has not evolved during q) appear at Fl = 0 such peaks can be a nuisance as they may obscure other wanted peaks. We will see below (section 9.5.6) that axial peaks can be suppressed with the aid of phase cycling, all be it at the cost of doubling the length of the phase cycle. 

The States-TPPI method does not suppress these axial peaks, but moves them to the edge of the spectrum so that they are less likely to obscure wanted peaks. All that is involved is that, each time td is incremented, both the phase of the pulse which precedes q and the receiver phase are advanced by 180° i.e. the... 

The nice feature of States-TPPI is that is moves the axial peaks out of the way without lengthening the phase cycle. It is therefore convenient to use in complex three- and four-dimensional spectra were phase cycling is at a premium. 

There are two additional points to make here. If the spins have not relaxed completely by the start of the sequence the initial magnetization will not be at equilibrium. Then, the above simplification does not apply. Secondly, the first pulse of a sequence is often cycled in order to suppress axial peaks in two-... 

A simple way of suppressing axial peaks is to select the pathway Ap = 1 on the first pulse this ensures that all signals arise from the first pulse. A two-step cycle in which the first pulse goes 0°, 180° and the receiver goes 0°, 180° selects Ap = 1. It may be that the other phase cycling used in the sequence will also reject axial peaks so that it is not necessary to add an explicit axial peak suppression steps. Adding a two-step cycle for axial peak suppression... 

An alternative is to cycle the last pulse to select the pathway Ap = -1, giving the cycle 0 1 2 3 for the pulse and 0 1 2 3 for the receiver. Once again, this does not discriminate against z-magnetization which recovers during the mixing time, so a two step phase cycle to select axial peaks needs to be added. 

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