Spectrum acquisition relaxation delay

It proved helpful for the purpose of noise reduction to perform relaxation experiments in an interleaved fashion, as one pseudo-3D experiment, where the 2D planes in the F2 dimension correspond to various relaxation delays. The acquisition order (3-2-1) is selected so that cycling through various relaxation delays (in R1 or R2 experiments) or through NOE/NONOE 2D planes is performed prior to incrementing the evolution period in the indirect dimension (FI) (see e.g. Ref. [16]). The resulting pseudo-3D spectrum can be processed as a set of 2D spectra in tl and t3 dimensions, and then analyzed in the usual way. This procedure reduces the noise arising from switching from one 2D experiment to the other and helps minimize temperature variations between the spectra acquired... 

A 100 MHz NMR spectrum of a mixture of ethanol (C2H6O) 5 18.3 (CH3), 5 57.8 (CH2) and bromoethane (C2HsBr) 5 19.5 (CH3) and 6 27.9 (CH2) in CDCI3 solution is given below. The spectrum was recorded with a long relaxation delay (300 seconds) between acquisitions and with the NOE suppressed. Estimate the relative proportions (mole %) of the 2 components from the peak intensities in the spectrum. 

Fig. 14. ID COSY-RELAY spectra of two terminal glucoses of oligosaccharide 5. (a) Partial H spectrum of 5 at 600 MHz and 27°C. Spectra (b) and (c) were acquired using the pulse sequence in fig. 13(a) (k = 3) with the initial polarization transfer from overlapping anomeric protons of terminal glucoses. Duration of the Gaussian pulse was 50 ms, to = 39 ms, T] = 50 ms, A = 9.09 ms, T2 = 50 ms, T3 = 40 ms, number of scans was 64, relaxation delay and acquisition times were 2 and 1.4 s, respectively. AT = 0 for the first and N = 1 for the second spectrum, (d) is the sum of (a) and (b), (e) is the difference between (a) and (b). (Reprinted with permission from ref. [38]. Copyright 1993 ESCOM Science Publisher...

The typical peak width with analytical columns of 4.6 mm i.d. and a 1 ml/min flow rate is of the order of 10-30 s. The acquisition of NMR spectra with a short relaxation delay and an acquisition time of below 1 s allows the acquisition of 8-24 transients for one spectrum during the presence of a peak in the NMR cell. This low number of transients limits the detectable amount of sample to 5-10 xg per compound. 

Continuous-flow 19F LC-NMR spectra were acquired for 16 transients using 60° pulses into 8192 data points over a spectral width of 11 364 Hz, giving an acquisition time of 0.36 s. A relaxation delay of 0.64 s was added to give a total acquisition time for each spectrum of 16 s. The data were multiplied by a line-broadening function of 3 Hz to improve the signal-to-noise ratio and zero-filled by a factor of two before Fourier transformation. The results are presented as a contour plot with 19F NMR chemical shift on the horizontal axis and chromatographic retention time on the vertical axis. 

For the continuous-flow measurements, the pseudo-2D spectrum was recorded with a spectral width of 9616 Hz and 64 transients with 8K complex data points, thus resulting in an acquisition time of 0.42 s/transient along the 128 t increments. A relaxation delay of 1.2 s was used and the time resolution... 

The standardized pulse program for a proton decoupled 13C spectrum is shown in Figure 4.2a. The sequence is relaxation delay (Rd) (see Section 4.2.3), rf pulse (6), and signal acquisition (t2). The proton channel has the decoupler on to remove the H—13C coupling, while a short, powerful rf pulse (of the order of a few microseconds) excites all the 13C nuclei simultaneously. Since the carrier frequency is slightly off resonance FID (free induction decay), for all the 13C frequencies, each 13C nucleus shows a FID, which is an exponentially decaying sine wave. 

Figure 4.2b is a presentation of the FID of the decoupled 13C NMR spectrum of cholesterol. Figure 4.2c is an expanded, small section of the FID from Figure 4.2b. The complex FID is the result of a number of overlapping sine-waves and interfering (beat) patterns. A series of repetitive pulses, signal acquisitions, and relaxation delays builds the signal. Fourier transform by the computer converts the accumulated FID (a time domain spectrum) to the decoupled, frequency-domain spectrum of cholesterol (at 150.9 MHz in CDC13). See Figure 4.1b.

The 99 data points shown in Figure 3.17 are part of a total FID of 8000 complex pairs (total number of data points NP = 16,000). Since a single data point takes 180 p.s (the dwell time) to acquire on average, 16,000 points require 16,000 x 180 p.s = 2,880,000 p.s or 2.88 s to acquire. This is called the acquisition time (Bruker AQ Varian AT), and it represents the time required to record the entire FID once. This is not the time required for the entire spectrum to be acquired, since it does not include the relaxation delay and the pulse width, and it does not take into account the number of times the whole sequence is repeated (i.e., the number of scans or transients). In general,... 

Aside from the obvious improvement in the s/n ratio, which arises in part from concentrating the sample from 145 to 40 pL as well as from having all of the sample in the rf coil rather than 70%, the ketone carbonyl resonance at 219.5 ppm was observed with reasonable signal intensity whereas its presence was questionable at best in the 3 mm micro-dual probe 13C spectrum shown in the top panel. The s/n ratios of the overnight acquisitions were 5.5 1 for the 3 mm micro-dual probe vs. 11 1 for the Nano-probe . There is no obvious reason why the carbonyl resonance was not observed in the 3 mm micro-dual 13C spectrum, as the relaxation delay between acquisitions was set identically in the two experiments. These data also suggest that on a per-transient basis, that the performance of the Nanoprobe is perhaps somewhat lower than that of the 3 mm micro-dual probe. This inference is based on the nearly 4-fold concentration difference between the two probe formats. Assuming probe efficiencies were identical, an s/n ratio approaching 22 1 should have been observed for the Nano-probe based on the concentration increase. 

Figure 2-13 Apparent phase errors due to signal-truncation effects, (a) The distorted spectrum caused by an acquisition time that is too short (DT = 0 s). (b) The distortion-free spectrum after the introduction of a relaxation delay time (DT = 1 s).

Fig. 1. H- - C CT-HSQC spectrum of a sample of 1.5mM Val, Leu, lie (51) methyl-protonated maltose-binding protein (MBP), 2mM /3-cyclodextrin, 20 mM sodium phosphate (pH 7.2), 3mM NaN(, 200pM EDTA, 0. 1 mg/ml Pefabloc, 1 /ig//d pepstatin and 10% D20 recorded at 37°C, on a Varian Unity-1- 500-MHz spectrometer. Acquisition times of 28 and 64 ms were employed (/, t2) along with a relaxation delay of 1.5 s, fora total measuring time of 3 h. (a) Aliphatic region of the H- - C correlation map of MBP, illustrating the selectivity of labelling. Small amounts of residual protonation are observed at the Cy positions of a number of Pro/Arg residues, the Cp positions of Asp and Ser (aliased) residues, and the Cy2 methyl positions of lie. In all cases, intensities of these cross-peaks are less than 10% of the methyl peaks, (b) Methyl region of the H- - C HSQC. Reproduced with permission from Kluwer Academic Publishers Goto et al.H...

The example of 2,3-dimethylbenzofuran will close this section. In this molecule, the quaternary (ipso) carbons have relaxation times that exceed 1 minute. As discussed in Section 4.7, to obtain a decent spectrum of this compound, it would be necessary to extend the data acquisition and delay periods so as to determine the entire spectrum of the molecule and see the carbons with high Ti values. 

The spectrum was acquired at 705 MHz with H decoupling using a 0.7s acquisition time, 3ps (20°) pulse width, 16 transients, and a 3s relaxation delay. The spectrum was acquired at 188.6 MHz with WALTZ-16... 

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