Sample preparation deuterated solvent

For 13C experiments, the sample is usually prepared by dissolving the compound to be investigated in a deuterated solvent, which is usually required for field/frequency stabilization. For calibration, a small amount of a reference compound (usually tetra-methylsilane) is added to the sample. 

A sample for NMR spectroscopy can be taken from several stages of the sample preparation path (see Chapter 9) (14-19-28). Preferably, this would be a 5-10-mL portion of extract or of aqueous or organic liquid. Common to all these solutions is the large molar excess of H in the solvent compared to the amount of 111 in the possible target chemicals. This yields an intense solvent (e.g. H20, CH2C12) resonance in the 111 NMR spectrum, making the trace analysis difficult or even impossible. A usual procedure to avoid this problem is to replace the protonated solvent (e.g. H20) with the corresponding deuterated solvent (D20). Deuter-ated solvents are also used for the field-frequency lock of the spectrometer. 

Alternatively, an HPLC filter unit may be used for the filtration. The sample vessel is rinsed with a suitable amount of (deuterated) solvent and the rinsing solutions are added to the sample in the NMR tube. The pH of the aqueous liquid sample is determined, and can be adjusted with DC1 and NaOD solution. For measurements carried out in a standard 5-mm (o.d.) NMR tube, about 0.6-0.8 mL of solvent is required, and for 10-mm tubes, about 3-4mL. If considered necessary, some of the extractions can be performed with deuterated solvents, which will simplify the sample preparation procedure. Related practical sample preparations can be found in the literature (10 12 14 29). 

Because NMR is a nondestructive method, the NMR samples can be used for the preparation of samples for other analytical techniques. (Note that in some cases the deuterated solvent may deuterate the chemicals of interest affecting their detection, for example by GC/MS.)... 

We have addressed in our laboratory several aspects of the more rapid and robust identification of resin-bound entities. Firstly, related to the question of sensitivity and sample preparation, how much of a resin do we really need to obtain workable spectra in a given time lapse of 1 minute, and what is the optimal sample preparation procedure Whereas we and others in the early stages prepared the sample by swelling the resin in a separate recipient, and transferring part of the slurry into the rotor by a specially machined spatula, we later found out that swelling the resin beads directly into the rotor led in a faster and more economic way (less resin and deuterated solvent is needed) to more reprodu-... 

Samples for solution NMR spectroscopy are generally prepared using deuterated solvents. One reason for this is that, were non-deuterated solvents to be used (e.g. CH3CI in place of CD3CI) for a H NMR spectroscopic experiment, the signals due to the solvent would swamp those due to the sample. Deuterated solvents are commercially available, typically with >99.5% H label incorporated. The remaining unlabelled compound provides a useful internal reference signal in the H NMR spectrum of the sample under study. 

For and NMR experiments, the samples must be prepared in totally or partially deuterated solvents to avoid the interference created by solvent signals. In most of the modem NMR instmments, the deuterium signal from the solvent is used by an NMR lock system to avoid fluctuation of the magnetic field strength. Recent advances in NMR provide spectra with high resolution and high signal-to-noise ratio. This enables one to take the spectrum of the sample solution at very low concentrations. 

Several factors must be considered for a particular biomacromolecular structure application that will affect the choice of spectroscopic methods. These include structural resolution necessary, chemical nature of biomacromolecule (protein, nucleic add, or glycan), amount/concentration of biopolymer available, sample preparation (solid or solution), solvents of interest, and desired structure information (secondary or tertiary structure). Structural resolution varies considerably for the various spectroscopic methods, with X-ray diffraction and NMR providing atomic resolution (high resolution) and ultraviolet (UV) absorption revealing merely information about the polarity of the chromophore s environment (low resolution). X-ray studies require crystals while NMR experiments prefer solutions in deuterated solvent. Solvent preferences can affect the choice of spectroscopic method as, for example, infrared (IR) encoimters strong interference from water, while optical rotatory dispersion (ORD) and circniar dichroism (CD) do not. Some of the commonly used spectroscopic methods in structural analyses of biomacromolecules will be discussed. 

Nuclear Magnetic Resonance Spectroscopy. Like IR spectroscopy, NMR spectroscopy requires little sample preparation, and provides extremely detailed information on the composition of many resins. The only limitation is that the sample must be soluble in a deuterated solvent (e.g., deuterated chloroform, tetrahydro-furan, dimethylformamide). Commercial pulse Fourier transform NMR spectrometers with superconducting magnets (field strength 4-14 Tesla) allow routine measurement of high-resolution H- and C-NMR spectra. Two-dimensional NMR techniques and other multipulse techniques (e.g., distortionless enhancement of polarization transfer, DEPT) can also be used [10.16]. These methods are employed to analyze complicated structures. C-NMR spectroscopy is particularly suitable for the qualitative analysis of individual resins in binders, quantiative evaluations are more readily obtained by H-NMR spectroscopy. Comprehensive information on NMR measurements and the assignment of the resonance lines are given in the literature, e.g., for branched polyesters [10.17], alkyd resins [10.18], polyacrylates [10.19], polyurethane elastomers [10.20], fatty acids [10.21], cycloaliphatic diisocyanates [10.22], and epoxy resins [10.23]. 

The experiments performed in the HR-MAS probe require special sample preparation as the resin has to be placed in 4-mm rotors instead of the normal NMR tube used for standard solution compounds. A few milligrams of resin are transferred into the 4-mm HR-MAS NMR rotor with a spatula. Some pL (<40pL) of deuterated solvent is added to the rotor until the beads are completely swollen in the solvent, making a gel-like solution. (Some resin or some more solvent can be added to the rotor until the gel stage is acquired.) Finally the rotors are closed manually by using standard caps. 

H NMR spectra were recorded on Briiker ACP 400 or DPX 400 spectrometers using deuterated solvents obtained from CEA or Aldrich. Polymerization kinetics, followed by NMR, were recorded using the Briiker built-in kinetics software. Molecular mass analyses were carried out by gel permeation (size exclusion) chromatography on a Polymer Laboratories system. THF was the eluent at 1.0 mL min with a PL-gel 5 jim (50 X 7.5 mm) guard column, two PL-gel 5 pm (300 x 7.5 mm) mixed-C columns with a refractive index detector. Samples were compared against narrow standards of poly(methyl methacrylate), A/p = 200 to 1.577 x 10 g mol , obtained from Polymer Laboratories, except for methyl methacrylate dimer, trimer, and tetramer which were prepared by catalytic chain transfer polymerization at the University of Warwick. 

Several barley and wheat kernels and flour samples were used in the work presented in this chapter. The details concerning the plant material can be found in the relevant papers. The samples were prepared for NMR analysis by placing the kernel part (25-40 mg) or flour (app. 14 mg) in a 50 pi zirconia rotor (4.0 mm o.d.) followed by adding deuterated solvent (D2O or dimethyl sulfoxide (DMSO)) directly into the rotor. 5.8 mM 3-(trimethylsilyl)propionic acid-d4 sodium salt (TSP-d4) was added as chemical shift reference. 

One important remaining question to be discussed in relation to sample preparation of HR MAS NMR is the choice of solvent and exposure time to the solvent. We found that especially mature dry seeds tend to absorb the added deuterated water (D2O) and exchange it with internal water (H2O). The result of this process is a poorer shimming and in turn increasingly lower quality spectra over time. In order to evaluate if another solvent with advantage could be used to replace D2O, wheat flour samples were measured using deuterated DMSO with h HR MAS NMR (Figure 4). DMSO was chosen, since most... 

Technique 26, Section 26.1, describes the technique for preparing samples for proton NMR. Much of what is described there also applies to carbon NMR. There are some differences, however, in determining a carbon spectrum. Fourier transform instruments require a deuterium signal to stabilize (lock) the field. Therefore, the solvents must contain deuterium. Deuterated chloroform, CDClg, is used most commonly for this purpose because of its relatively low cost. Other deuterated solvents may also be used. 

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