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many pharmaceutical laboratories.52 Two approaches have been adopted to increase the throughput of LC-MS analysis: fast LC-MS,53,54 which utilizes a fast (2 3 min) chromatographic separation step, and parallel LC-MS,32,55,56 in which multiple HPLC columns are coupled to a single mass spectrometer. With these developments, LC-MS has become more suitable for combinatorial library analysis. In addition to FIA-MS and LC-MS, supercritical uid chromatography (SFC)-MS57,58 and CE-MS59,60,61 have also been adopted for combinatorial library analysis. SFC has a higher chromatographic resolution and faster separation speed than HPLC, and CE is a particularly useful in separating for ionic compound and chiral mixtures. However, mass spectrometry encounters problems when compounds with the same molecular weight are present. These compounds could be stereo-isomers, positional isomers, or simply molecules that happen to have the same molecular weights. This situation often occurs in tightly focused or closely related combinatorial libraries. To solve this ambiguity, NMR spectroscopy is usually employed to determine the structures of the compounds involved. NMR spectroscopy is a very powerful and widely adopted analytical tool for structural and quantitative determinations in chemistry, biology, material science, and many other areas. It is also a nondestructive technique and capable of deriving information on molecular dynamics. But traditional NMR spectroscopy suffers from low sensitivity and slow analysis speed; thus it is not an ideal tool to analyze the large number of compounds present in the combinatorial libraries. To meet the new challenges emerging from combinatorial chemistry and parallel synthesis methods, efforts are being made to make NMR more suitable for analyzing vast number of samples quickly, and a number of these developments are described below. 7.1.2. High-Throughput Serial NMR Analysis
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A number of factors contribute to the slow speed of NMR analysis, including sample preparation, sample loading/unloading, shimming to ensure high-resolution spectra, frequency locking, as well as data acquisition and analysis time. To increase the speed of NMR analysis, the most straightforward way is to reduce the time needed for preparing, loading, and retrieving the NMR samples. Automatic sample-changing mechanisms have been integrated with NMR spectrometers for at least 10 years.62 Such systems include a sample holder tray in which a large numbers of samples are placed; a mechanism to track each sample; and a robotic device to load the samples into standard 3 mm, 5 mm, or 10 mm NMR tubes, and to move sample tubes pneumatically into and out of the magnet. Many NMR labs, such as those in University of Minnesota, University of California at
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Los Angeles, Conoco Research, and IBM, started to use automatic sample changers to speed up NMR analysis in late 1980s, with throughput rates of up to 100 samples per day on a single NMR spectrometer.62 In 1988 Abbott Laboratories developed their rst automation systems to achieve totally automated NMR analysis, and both the turnaround time and the cost per sample were lowered signi cantly.63 For example, the number of NMR samples run per year at Abbott had grown to nearly 60,000 in 1995 upon the implementation of three totally automated NMR systems. Currently automatic sample changers compatible with different NMR systems are provided by the major NMR system manufacturers, including Bruker, Varian, and JEOL. These commercially available automatic sample changers are capable of carrying out tasks such as recording the sample identi cation number, preparing sample solution, pipetting solution into a clean NMR tube, inserting an NMR tube into the magnet, and then removing the NMR tube and disposing of the sample when the NMR experiment is nished. Currently the use of NMR systems equipped with automatic sample changers has become routine for many NMR laboratories with high-volume needs. Conventional high-resolution NMR spectroscopy requires the use of precision glass NMR tubes, which are delicate and expensive. Sample preparation involves lling NMR tubes, loading tubes into the magnet one at a time, and removing each tube from magnet after the NMR experiments are nished. Although the use of robotic sample changers has made the automation of the sample preparation and changing possible, the use of NMR tubes limits its speed and ef ciency for high-throughput applications. To avoid the steps of transferring samples from a reservoir into NMR tubes and thus further increasing the throughput, an alternative solution is to use a ow NMR probe.28,64 Such ow probes, which are also known as tubeless NMR probes, have a permanently mounted sample chamber or ow cell, on which an RF detection coil is usually wrapped. The sample is introduced into the ow cell, and a rinsing step is necessary to eliminate the carryover from the previous sample. Because the RF coil is mounted closer to the sample, an increased lling factor is obtained. Therefore ow NMR probes usually have higher sensitivity than conventional NMR probes. However, because of the permanent mounting of the RF coil on the ow cell, it is not possible to spin the sample as is done with normal NMR tubes. Therefore the resolution of ow NMR probes is typically poorer. Standard ow NMR probes have detection volumes that range from 60 to 250 mL,65,66,67,68 which are smaller volumes than those used in conventional 5-mm NMR probes. Recent developments in microcoil probe technologies have pushed the sample volume requirements in ow NMR to the nanoliter to few microliter range.25
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