Sample Requirements for NMR

The ability of NMR to provide information regarding the specific bonding structure and stereochemistry of molecules of pharmaceutical interest has made it a powerful analytical tool for structure elucidation (see Chapter 12). Unfortunately, NMR has traditionally been sensitivity-limited compared to other analytical techniques. Conventional sample requirements for NMR are on the order of 10 mg, as compared with mass spectroscopy, for example, which requires less than 1 mg. Therefore, NMR spectroscopy historically has not been the first choice for an analytical chemist when identifying an unknown compound. 

The size of sample required has been reduced by a number of technical developments including micro inverse probes and micro cells (references in Martin et al. 1998), and has been reduced even further using a newly developed 1.7-mm submicro inverse-detection gradient probe (Martin et al. 1998). The combined use of inverse detection probes with solenoid microcoils has also been developed to reduce sample volumes for NMR (Subramanian and Webb 1998). 

Table F1.4.2 NMR Sample Tube Requirements for NMR Instruments with Different Field Strengths"...

The injection of the sample into the LC-NMR system can be carried out by an autosampler or a manual injection valve. The only difference which has to be considered is the fact that the amount of sample required for the NMR system is larger than that required for UV detection. Therefore, it is often necessary to inject sample volumes which exceed 100 jjlL In such cases, it is mandatory to dissolve the sample in the starting solvent phase in order to avoid additional LC gradients created by the sample solvent being injected. 

Another consideration is the amount of sample (in mg) required for NMR. Because it is the concentration (mM) of molecules that determines the signal strength in NMR, as the molecular size increases the desired concentration of around 1 mM corresponds to larger and larger amounts of protein (Table 12.1). For a small protein (e.g., RNase at 12.6 kD) the sample size is around 6 mg. Talk to a biochemist or molecular biologist if you think this is a small amount of pure protein Now move to chymotrypsin (22.6 kD), which would require 11 mg of pure protein to be soluble and monomeric in 0.5 mL of water. For fatty acid synthase, with 20,000 amino acid residues and 21 polypeptide chains, we would need to dissolve 1.15 g of protein in our 0.5 mL sample volume, clearly an impossibility. 

Stop-flow requires the calibration of the delay time, which is the time required for the sample to travel from the UV detector to the NMR flow cell, which depends, in turn, on the flow rate and the length of the tubing connecting HPLC with NMR. Because the chromatographic run is automatically stopped when the chromatographic peak of interest is in the flow cell, the amount of sample required for the analysis can be reduced and 2-D NMR experiments, such as correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and others, can be obtained because the sample can remain inside the flow cell for days. It is possible to obtain NMR... 

Shortcuts should not be taken in isolation efforts for NMR analysis. An analytical HPLC cleanup/desalting is required for NMR analysis. This improves the sample purity and quality of NMR data. It is especially difficult to characterize dimers and oxidations of heteroatoms if a sample is not of high purity. 

An advantage of IR spectroscopy in general, and as applied to humic substances in particular, is the small quantity of sample required. For example, the alkali halide pressed-pellet technique, as normally carried out, requires about 1-2 mg of sample, which is considerably less than that required for the other most useful spectroscopic method, NMR, in the study of humic substances (see Chapter 22). However, microsampling methods have been developed which can record IR spectra on less than 0.01 /xg of sample (Alpert et al., 1964 Parker, 1971 Price, 1972 Griffiths and Block, 1973). These special techniques may have advantage in the study of humic samples isolated only in minute quantities, or for investigating small samples obtained in the fractionation of humic substances. 

The sample size requirement for NMR depends on the instrumentation available. High magnetic fields such as those on 600-800 MHz spectrometers provides optimal resolution and serrsitivity and allow effective 2D NMR molecular structural characterization of samples as small as 1 mM Fd in 0.5 ml in H2O. The relatively small size of Fds, howev , do not present serious resolution problems 0 that similarly effective 2D NMR studies can be carried out on lower-field spec-... 

Other forensic samples require different methods of preparation. Biological fluids often may be analyzed directly, or after the addition of a small amount of D2O for signal frequency locking. Plant material, tissue samples, and biological fluids may be put through extraction procedures to obtain samples suitable for NMR analysis. Explosives and accelerant samples may be prepared by normal chemical procedures. Postexplosion residues and fire debris may be extracted, and samples from these extracts prepared in the normal way. 

Sample preparation for NMR is essentially the same as for NMR spectroscopy except that significantly more material is required to obtain a NMR spectrum in a reasonable amount of time. In this case, acceptable signal-to-noise levels can be obtained on 40 mg of material in about 10 min. 

---