Use of Microcoil Probes to Acquire More Sensitive NMR Spectral Data

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical technique that is commonly used to provide structural information as well as other chemical metrics such as sample composition and highly involved reaction kinetics. While NMR is a very informative tool, its sensitivity is orders of magnitude lower than other analytical detection techniques (e.g., mass spectrometry or UV detection). A number of approaches have been developed to improve the signal-to-noise ratio of an NMR measurement (i.e., high-strength magnets and cryogenically cooled probes). These approaches do improve the S/N, but they also lead to a significant increase in instrument cost. An alternative way to enhance the sensitivity is to increase the sample size, but this is not an acceptable approach for many studies due to the difficulty of obtaining the sample (especially for molecules of biological interest). In recent years, the use of microcoil technology has dramatically improved the sensitivity of NMR measurements at a considerably lower cost than cryogenic probes.1

Delivery of the sample to the microcoil probe is an important issue that is closely related to the improvement in sensitivity, since both factors must be optimized to maximize the throughput of the system and its utility to the analyst. In many facilities, a high-throughput autosampler is employed, while chromatographic or electrophoretic methods of separation are used in other facilities. A seamless analytical method is desired to optimize the throughput of the analytical laboratory.

This paper describes the use of microcoil technology to improve the sensitivity of NMR measurements. It discusses a number of sample handling approaches ranging from microflow injection to solid-phase extraction (SPE), HPLC, and electrophoretic migration.

Use of a microcoil to optimize the sensitivity of an NMR spectrometer for analysis of microgram and submicrogram mass levels

In general, the radio frequency (RF) receiver coil should closely conform to the sample to ensure good detection sensitivity. A properly designed NMR probe will maximize both the observe factor, which is the ratio of the sample volume being observed by the RF coil to the total sample volume required for analysis, and the fill factor, the ratio of the sample volume being observed by the RF coil to the coil volume. Since the sensitivity of the coil is roughly inversely proportional to the coil size, a probe that employs a 1-mm RF coil has a mass sensitivity that is roughly tenfold greater than a conventional tube probe that utilizes RF coils closer to 10 mm in size. Due to their small size and patented approach to magnetic susceptibility matching, CapNMR microcoil probes (Protasis, Inc., Marlboro, MA) are easy to use and shim and typically require only a handful of the available system shims to meet resolution specifications. A detailed discussion of microcoil NMR design is presented in Ref. 1.

Figure 1 - NMR analysis by cryoprobe and microcoil probe. a) Single scan using a cryoprobe. The S/N was normalized over the sample quantity in the active volume (230 μL, Vmrm = 1.5 μL). b) Scan using a microcoil probe. Reproduced with permission from Dr. Feng Xu, Bristol-Myers Squibb Research Institute (Wallingford, CT).

An example of the utility of a microprobe is shown in Figure 1, in which the spectrum of an unknown is presented using a CapNMR probe and a cryoprobe. While the S/N for the two spectra are similar, it is important to note that the spectrum obtained from the CapNMR probe had considerably less background signal effects due to the 100-fold reduction in solvent volume for the mass detected.

Use of a microcoil to obtain NMR data from biomolecules

The use of a microcoil may be advantageous for the collection of NMR spectra of biomolecules since obtaining a sufficiently large quantity of a protein (or other large molecule) may be exceedingly difficult or tedious. In a recent study, Peti et al.2 used the TXI HCN z-grad CapNMR probe to determine protein folding, as well as for complete sequence-specific backbone assignment of a 10-kD protein (TM0979 from Thermotoga maritima). The probe is well suited for obtaining the NMR spectrum of protein samples produced in nonoptimized, highly automated protein expression pipelines (where the amounts of protein produced routinely have not been sufficient for traditional 5-mm tube NMR investigations). The probe opens up a new avenue for protein folding screening in the context of structural proteomic projects.

Peti and coworkers have demonstrated sequence-specific backbone assignment using heteronuclear triple resonance HNCA and HNCOCA experiments on the CapNMR probe. The group has also demonstrated TOCSY transfer between the aliphatic region and the aromatic region for the first time in an HCCH–TOCSY experiment for purposes of side-chain assignments. This measurement was possible because of the low power requirement of the CapNMR probe; it is a direct result of the mass sensitivity of the probe and is precluded in many larger probes due to RF power restrictions.

Automation and sample handling

High throughput is an increasingly critical consideration in many laboratories in which a large number of samples are delivered to the NMR probe via an autosampler. The overall time per sample is a critical issue and is determined by the residence time of the sample in the probe and the sample preparation and delivery steps. When the microcoil probe is employed, the sensitivity of the measurement is increased; thus, the residence time for data acquisition can be reduced. Additional time can be shaved off the time per sample by optimizing each of the additional steps in the sample preparation and transfer process.

One of the most widely used NMR automation systems employed in NMR flow injection systems is the model 215 liquid handler (Gilson, Middleton, WI). Protasis and MRM (Savoy, IL) have developed a means of loading samples into the CapNMR probe using this liquid handler in conjunction with a high pressure, HPLC-grade pump. This has convinced a number of major pharmaceutical companies to adapt their flow NMR system to the CapNMR probe.

Figure 2 - Automated flow injection for capillary NMR. Reproduced with permission from Dr. Timothy Peck, Protasis/MRM Corp.

Scientists at GlaxoSmithKline (Research Triangle Park, NC) have advanced this development by implementing a filter maintenance protocol that provides for significantly increased mean time to failure. They have reported error-free injection of thousands of samples without filter change. The Inova 600-MHz NMR spectrometer based system (Varian, Palo Alto, CA) that was used in this study included a specialized needle (Gilson), custom loading port (Protasis/MRM), filter, and CapNMR probe. Figure 2 shows a collection of spectra from a 96-well plate. This capillary flow automation system requires considerably less sample and solvent and provides less system downtime and smaller solvent residual peaks.

The Open Access Automation NMR system (Protasis/MRM) comprises a user interface to control the CTC autosampler (Leap Technologies, Carrboro, NC) with the NMR. It can be used with all commercial spectrometers and provides an operator-centric view of the system for sample control, report management, and accounting information. The liquid handler is specifically designed for microliter volume samples.

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