Small sample NMR

For on-bead analysis vibrational spectroscopy (IR-spectroscopy) can be employed attenuated total reflection is a method allowing fast and nondestructive on-bead analysis of small samples (single bead analysis) without significant sample preparation. Solid phase NMR is the method of choice if complex structural analysis is intended on the support. Spatially resolved analysis on the resin is possible with microscopic techniques. 

NMR spectrometers have improved significantly, particularly in the present decade, with the development of very stable superconducting magnets and of minicomputers that allow measurements over long time periods under homogeneous field conditions. Repetitive scanning and signal accumulation allow H-NMR spectra to be obtained with very small sample quantities. 

Rycroft, D. S. 1996. Fingerprinting of plant extract using NMR spectroscopy application to small samples of liverworts. Chem. Comm. (18) 2187-2188. 

Flow NMR has recently been eclipsed by the advent of robotic sample handling systems capable of dealing with very small sample quantities and volumes. We now have a system operating in our laboratory that makes up samples directly into 1 mm NMR tubes, using only about 8 ul of solvent. These can be run under automation and the tubes emptied back into the plate wells by the same robot. This technology offers superior performance and largely gets around the problems of contamination and recovery. 

A small sample of the hydride is suspended in benzene-dg and assayed in a 5-mm NMR tube by treatment with a known amount of excess acetone (Equation 1). The relative areas of the signal for the mono- and dlisopropoxides are determined by 1H NMR (300 MHz, C D6 integrating the methyl doublets) Cp2Zr(H)CI 94-96%, Cp2ZrH2 4-6% 2a... 

Smaller diameter probes reduce sample volumes from 500 to 600 pi typical with a 5 mm probe down to 120-160 pi with a 3 mm tube. By reducing the sample volume, the relative concentration of the sample can be correspondingly increased for non-solubility limited samples. This dramatically reduces data acquisition times when more abundant samples are available or sample quantity requirements when dealing with scarce samples. At present, the smallest commercially available NMR tubes have a diameter of 1.0 mm and allow the acquisition of heteronuclear shift correlation experiments on samples as small as 1 pg of material, for example in the case of the small drug molecule, ibu-profen [5]. In addition to conventional tube-based NMR probes, there are also a number of other types of small volume NMR probes and flow probes commercially available [6]. Here again, the primary application of these probes is the reduction of sample requirements to facilitate the structural characterization of mass limited samples. Overall, many probe options are available to optimize the NMR hardware configuration for the type and amount of sample, its solubility, the nucleus to be detected as well as the type and number of experiments to be run. 

While microscopic techniques like PFG NMR and QENS measure diffusion paths that are no longer than dimensions of individual crystallites, macroscopic measurements like zero length column (ZLC) and Fourrier Transform infrared (FTIR) cover beds of zeolite crystals [18, 23]. In the case of the popular ZLC technique, desorption rate is measured from a small sample (thin layer, placed between two porous sinter discs) of previously equilibrated adsorbent subjected to a step change in the partial pressure of the sorbate. The slope of the semi-log plot of sorbate concentration versus time under an inert carrier stream then gives D/R. Provided micropore resistance dominates all other mass transfer resistances, D becomes equal to intracrystalline diffusivity while R is the crystal radius. It has been reported that the presence of other mass transfer resistances have been the most common cause of the discrepancies among intracrystaUine diffusivities measured by various techniques [18]. 

To overcome the problem of sample availability, smaller volume NMR probes, nanoprobes , have been introduced for structure elucidations of samples that were previously considered too small for NMR analysis. 43 ... 

Compared with chiroptical methods and nuclear magnetic resonance spectroscopy (NMR), only chiral chromatography by direct and indirect methods is suitable for the accurate determination of enantiomeric impurities of less than 1% and for quantitative stereochemical analyses of small sample amounts (for example, in vivo studies of the metabolic pathway or pharmacokinetic effects of chiral pharmaceuticals.)... 

Inverse-detected experiments have had the greatest effect in making 15N NMR experiments feasible for small samples. These experiments take advantage of the higher sensitivity of NMR to facilitate the observation of insensitive nuclei like 13C and 15N. The H-13C heteronuclear multiple quantum coherence (HMQC) and the related heteronuclear multiple-bond correlation (HMBC) experiments are important in contemporary natural products... 

Mass spectrometry and high-resolution NMR spectroscopy, applicable to small samples of carbohydrate, yield essential information about sequence, configuration at anomeric and other carbons, and positions of glycosidic bonds. 

Because the characterization of support-bound intermediates is difficult (see below), solid-phase reactions are most conveniently monitored by cleaving the intermediates from the support and analyzing them in solution. Depending on the loading, 5-20 mg of support will usually deliver sufficient material for analysis by HPLC, LC-MS, and NMR, and enable assessment of the outcome of a reaction. Analytical tools that are particularly well suited for the rapid analysis of small samples resulting from solid-phase synthesis include MALDI-TOF MS [3-5], ion-spray MS [6-8], and tandem MS [9]. MALDI-TOF MS can even be used to analyze the product cleaved from a single bead [5], and is therefore well suited to the identification of products synthesized by the mix-and-split method (Section 1.2). The analysis and quantification of small amounts of product can be further facilitated by using supports with two linkers, which enable either release of the desired product or release of the product covalently bound to a dye [10-13], to an isotopic label [11], or to a sensitizer for mass spectrometry [6,14,15] (e.g., product-linker-dye- analytical linker -Pol). 

The remarkable versatility of NMR as an analytical method has diminished the importance of IR analysis in modern laboratories but it remains a very useful technique. The very small amount of sample required and the prominence of functional group absorption means that the progress of a reaction can be monitored very conveniently. For example, the reduction of an aldehyde to an alcohol will be accompanied by the disappearance of the prominent C=0 peak and the appearance of a C—OH absorption. Because both peaks are so readily identifiable in a small sample, the reaction is easy to follow and its completeness can be assayed. 

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