Relaxation reagents

It is important to avoid saturation of the signal during pulse width calibration. The Bloch equations predict that a delay of 5 1] will be required for complete restoration to the equilibrium state. It is therefore advisable to determine the 1] values an approximate determination may be made quickly by using the inversion-recovery sequence (see next paragraph). The protons of the sample on which the pulse widths are being determined should have relaxation times of less than a second, to avoid unnecessary delays in pulse width calibration. If the sample has protons with longer relaxation times, then it may be advisable to add a small quantity of a relaxation reagent, such as Cr(acac) or Gkl(FOD)3, to induce the nuclei to relax more quickly. 

Figure I. 39.7 MHz Si NMR spectra of PSQ and APSQ obtained from PSQ-B in acetone-d6. Chromium acetylacetonate was used as a relaxation reagent, and transients were 5000. PSQ-A (Mw = 900) and PSQ-B (Mw = 9500) were purchased from Owens-Illinois and Petrarch Systems, respectively.

The steady-state free-procession (SSFP) technique was used with no NOE, and in the case of aniline the T of the 15N was reduced by addition of chromium acetylacetate as a relaxation reagent to the sample. The 15N spectrum of aniline showed a single broad line (Avi/2 = 42 Hz) in which, because of proton exchange125,126, there is no evidence of N H coupling (J = 80 Hz). 

All of the polymers have resonances at unusually high field (>3.0 X) suggesting that some of the protons are held in close proximity to adjacent aromatic rings. This possibility is further supported by the observation that relaxation reagents selectively collapse the lowest field peak, which is expected to be the resonance of protons least encumbered by neighboring aromatic rings and therefore more accessible to the relaxation reagent. 

Fig. 6. Selected spectral regions of a NOESY spectrum of BPTI recorded with the pulse sequence of fig, 5(A), except that the first spin-lock pulse was omitted and a Bo gradient was applied during the NOESY mixing time. Protein concentration 20 mM in 90% H2O / 10% D2O, pH 6.9, 36°C. The relaxation reagent GdDTPA-BMA was added at a concentration of 750 pM to enhance the relaxation of the water protons. Spin-lock pulse 2 ms, rm(NOE) = 50 ms, r = 190 ps. Positive and negative levels were plotted without distinction. The arrow identifies the cross section containing the intermolecular water-protein cross peaks. (Reproduced by permission of the American Chemical...

Quantitative measurements in NMR are based on the area of the signals present in the spectrum. Signal areas can be produced as numerical values proportional to the area or, on less modern instruments, from the integration plots that are superimposed on the spectrum (Fig. 9.1). For the proton, the precision obtained in area measurements does not exceed l % even if continuous wave instruments are used at slow scanning speeds. In l3C NMR, it is preferable to add a relaxation reagent in order to avoid saturation related to relaxation times that alter the intensity of the signal. Using the molar ratios that are easily accessible from the spectrum, it is possible to deduce concentrations. 

Quaternary carbon nuclei e.g. CO carbons in metal carbonyls) display particularly large spin-lattice relaxation times, so that relaxation reagents such as chromium acetyl-ace tonate must be added to the sample solution in order to obtain sufficient signal noise within reasonable accumulation times. 

Using single-frequency and noise-modulated resonance and off-resonance proton decoupling, 7] relaxation time measurements, relaxation reagents like Gd (fod)3 and specifically deuterated compounds, all the carbons in retinal isomers, the model compounds a-and /i-ionone, and vitamin A and its isomers [165, 555-557] were assigned. The olefinic ring carbons (C-5 and C-6) could be identified on the assumption that the 13C relaxation times are dominated by intramolecular dipole-dipole interactions with neighboring protons and that the same rotational correlation time characterizes the interactions for both carbons. Consequently the ratio of T/s for C-5 and C-6 can be estimated from eq. (5.1)... 

The study of nucleic acid bases by NMR has been reported in a number of monographs (/), but very little data is available on the, 3C and, 5N NMR chemical shift tensors in these compounds. The low sensitivity of NMR spectroscopy and the long relaxation times exhibited by many of these compounds have posed the main impediments for these studies. The use of sample doping with free radical relaxation reagents, to reduce the relaxation times facilitating 2D multiple pulse experiment (2, 3), enables one to measure and analyze the principal values of the chemical shift tensors in natural abundance samples. In previous papers from this laboratory we have presented, 5N NMR chemical shift principal values for adenine, guanine, cytosine, thymine and uracil (4, 5). 

Aminomethylbenzylphenylmethylsilane, chiral at the silicon center, has been used as an NMR shift and relaxation reagent.313 Both enantiomers are obtained from the racemic material by fractional crystallization of the (+)-tartaric acid salts.314... 

Quantitative NMR measurements always require a number of precautions159. The well-known unfavourable properties of silicon nuclei are a further aggravation. Quantitative measurements of 29Si NMR require the use of IGD to eliminate NOE, long delays between scans (5-10 times the longest T in the sample) and/or addition of a relaxation reagent [e.g. chromium or iron triacetylacetonate, Cr(acac)3 or Fe(acac)3], to enhance relaxation... 

A different study of the properties of the lipid layer can be made by incorporating organic shift or relaxation reagents, Ln(III)(FOD)3, in the membrane53) (Fig. 13). It is then possible to study either the partitioning of molecules into the bilayer, e.g. CHC13, or the binding of say proteins to the bilayer surface. While we have seen both these effects we have not yet studied them in detail but hopefully this procedure will be used to find the special parts of proteins which bind to membranes. 

Addition of a paramagnetic relaxation agent enables quantitative determination of carbons by C NMR. This technique has been applied to a series of phenylindoles using chromium(lll) acetylacetonate (Cr(acac)3) as the relaxation reagent <1996M111>. 

The study of the fiillerenes by C NMR spectroscopy is not straightforward. C has a low natural isotopic abundance (1.11%) and a low relative receptivity. In addition, the carbon atoms in fiillerenes have relatively long relaxation times and spectra for the higher, less symmetric fiillerenes can only be obtained within a reasonable time by using paramagnetic relaxation reagents, typically Cr(acac)3. 

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