NMR detection with multiple solenoidal microcoils for continuous-flow capillary electrophoresis

Nuclear magnetic resonance (NMR) spectroscopy represents a promising on-line detector for capillary electrophoresis (CE). The inherent poor sensitivity of NMR mandates the use of NMR probes with the highest mass sensitivity, such as those containing solenoidal microcoils, for CE/NMR hyphenation. However, electrophoretic current degrades the resolution of NMR spectra obtained from solenoidal coils. A new method to avoid microcoil NMR spectral degradation during continuous-flow CE is demonstrated using a unique multiple solenoidal coil NMR probe. The electrophoretic flow from a single separation capillary is split into multiple outlets, each possessing its own NMR detection coil. While the CE electrophoretic flow is directed through one outlet, stopped-flow, high-resolution NMR spectra are obtained from the coil at the other outlet. The electrophoretic flow and NMR measurements are cycled between the outlets to allow a continuous CE separation with "stopped-flow" detection. As a new approach for improving multiple coil probe performance, the magnetic field homogeneity is automatically adjusted (via the shim coils of the magnet) for the active coil. The multiple microcoil CE/NMR coupling has been used to analyze a <3 nmole mixture of amines while obtaining between 1 and 2 Hz line width, demonstrating the ability to avoid electrophoretic current-induced line broadening.     

Capillary isotachophoresis/NMR: extension to trace impurity analysis and improved instrumental coupling

Building upon its promising initial performance, the online coupling of capillary isotachophoresis (cITP) to nuclear magnetic resonance (NMR) is extended to trace impurity analysis. By simultaneously concentrating and separating dilute charged species on the basis of their electrophoretic mobility, cITP greatly facilitates NMR structural elucidation. cITP/NMR appears particularly attractive for identifying trace charged synthetic and natural organic compounds obscured by large excesses of other components. A 9.4 microL injection of 200 microM (1.9 nmol) atenolol in a 1000-fold excess of sucrose (200 mM) is analyzed by cITP/NMR. A microcoil, the most mass sensitive NMR probe, serves as the detector as it provides optimal NMR observation of the capillary-scale separation. cITP successfully isolates the atenolol from the sucrose while concentrating it 200-fold to 40 mM before presentation to the 30 nL observe volume microcoil, thereby enabling rapid 1H NMR spectral acquisition of atenolol (experimental time of 10 s) without obstruction from sucrose. For this particular probe and sample, the stacking efficiency is near the theoretical limit as 67% of the sample occupies the 1 mm long microcoil during peak maximum. A multiple-coil probe with two serial 1 mm long microcoils arranged 1 cm apart has been developed to facilitate peak trapping and sample band positioning during cITP/NMR.     

Portable microcoil NMR detection coupled to capillary electrophoresis

High-efficiency separation techniques, such as capillary electrophoresis (CE), coupled to a nondestructive nuclear magnetic resonance (NMR) spectrometer offer the ability to separate, chemically identify, and provide structural information on analytes in small sample volumes. Previous CE-NMR coupled systems utilized laboratory-scale NMR magnets and spectrometers, which require very long separation capillaries. New technological developments in electronics have reduced the size of the NMR system, and small 1-2 T permanent magnets provide the possibilities of a truly portable NMR. The microcoils used in portable and laboratory-scale NMR may offer the advantage of improved mass sensitivity because the limit of detection (LOD) is proportional to the coil diameter. In this work, CE is coupled with a portable, briefcase-sized NMR system that incorporates a microcoil probe and a 1.8 T permanent magnet to measure (19)F NMR spectra. Separations of fluorinated molecules are demonstrated with stopped- and continuous-flow NMR detection. The results demonstrate that coupling CE to a portable NMR instrument is feasible and can provide a low-cost method to obtain structural information on microliter samples. An LOD of 31.8 nmol for perfluorotributylamine with a resolution of 4 ppm has been achieved with this system.     

Effect of magnetic field strength on NMR-based metabonomic human urine data. Comparative study of 250, 400, 500, and 800 MHz

Metabonomic analysis of urine utilizing high-resolution NMR spectroscopy and chemometric techniques has proven valuable in characterizing the biochemical response to an intervention. To assess the effect of magnetic field strength on information contained in NMR-based metabonomic data sets, 1H NMR spectra were acquired on 250-, 400-, 500-, and 800-MHz instruments, respectively, on the same set of human urine samples collected before and after dietary interventions with milk and with meat proteins. Partial least-squares regression discriminant analyses (PLS-DA) were performed in order to elucidate the ability of the 1H spectra acquired at various field strengths to identify possible spectral differences and discriminate between pre- and postintervention samples. The loadings from PLS-DA contained the same spectral regions, implying that the same metabolites were involved in the discrimination independent of magnetic field strength. The investigation revealed a strong increase in prediction performance and thereby spectral information content when increasing the magnetic field strength from 250 to 500 MHz, while from 500 to 800 MHz the increase was less pronounced.     

Nanoliter-volume 1H NMR detection using periodic stopped-flow capillary electrophoresis.

Recent advances in the analysis of nanoliter volumes using 1H NMR microcoils have led to the application of microcoils as detectors for capillary electrophoresis (CE). Custom NMR probes consisting of 1-mm-long solenoidal microcoils are fabricated from 50-micron diameter wire wrapped around capillaries to create nanoliter-volume detection cells. For geometries in which the capillary and static magnetic field are not parallel, the electrophoretic current induces a magnetic field gradient which degrades the spectroscopic information obtainable from CE/NMR. To reduce this effect and allow longer analyte observation times, the electrophoretic voltage is periodically interrupted so that 1-min high-resolution NMR spectra are obtained for every 15 s of applied voltage. The limits of detection (LODs; based on S/N = 3) for CE/NMR for arginine are 57 ng (330 pmol; 31 mM) and for triethylamine (TEA) are 9 ng (88 pmol; 11 mM). Field-amplified stacking is used for sample preconcentration. As one example, a 290-nL injection of a mixture of arginine and TEA both at 50 mM (15 nmol of each injected) is stacked severalfold for improved concentration LODs while achieving a separation efficiency greater than 50,000. Dissolving a sample in a mixture of 10% H2O/90% D2O allows H2O to serve as the nearly ideal neutral tracer and allows direct observation of the parabolic and flat flow profiles associated with gravimetric and electrokinetic injection, respectively. The unique capabilities of CE and the rich spectral information provided by NMR spectroscopy combine to yield a valuable analytical tool, especially in the study of mass-limited samples.     

Microflow NMR: concepts and capabilities

The principles and parameters to consider when choosing an NMR probe for analysis of a volume- or mass-limited sample are identified and discussed. In particular, a capillary-based microflow probe is described which has a mass sensitivity comparable to cryoprobes (observe volume approximately 40 microL), but with several distinct advantages. The microflow probe has a flowcell volume of 5 microL and an observe volume of 1.5 microL and is equipped with proton and carbon observe channels, deuterium lock, and z-gradient capability. The entire flow path is fused silica; inlet and outlet capillary inner diameters are 50 microm to minimize sample dispersion, making it well-suited to volume-limited samples. An injected sample of 1 nmol of sucrose (0.34 microg in 3 microL, 0.33 mM; MW = 342 g/mol) yields a 1D proton spectrum in 10 min on a spectrometer of 500 MHz or higher. In another example, 15 microg of sucrose (in 3 microL; 15 mM, 45 nmol) is injected and parked in the probe to yield a heteronuclear multiple-quantum coherence (HMQC) spectrum in less than 15 h. The natural product muristerone A (75 microg in 3 microL, 50 mM, 150 nmol; MW = 497 g/mol) was delivered to the flow cell, and a gradient correlation spectroscopy spectrum was acquired in 7 min, a gradient HMQC in 4 h, and a gradient heteronuclear multiple-bond correlation in 11 h. Four basic modes of sample injection into the probe vary in degree of user intervention, speed, solvent consumption, and sample delivery efficiency. Manual, manual-assisted (employing a micropump), automated (using an autosampler), and capillary HPLC modes of operation are described.     

High-resolution capillary tube NMR. A miniaturized 5-microL high-sensitivity TXI probe for mass-limited samples,off-line LC NMR,and HT NMR

A new triple-resonance (TXI) (1H, 13C, 15N) high-resolution nuclear magnetic resonance (NMR) capillary probe with 2.5-microL NMR-active sample volume (V(obs)) was built and tested for applications with mass- and volume-limited samples and for coupling of microbore liquid chromatography to NMR. This is the first microliter probe with optimized coil geometry for use with individual capillary tubes with an outer diameter of 1 mm. The 90 degree pulse lengths of the 1-mm microliter probe were below 2 micros for proton, below 8 micros for carbon, and below 20 micros for nitrogen, and a spectral line width at signal half-height below 1 Hz was obtained. Compared to a conventional 5-mm probe, the new 600-MHz 1-mm TXI microliter probe with z-gradient shows an increase in mass sensitivity by a factor of 5, corresponding to a 25-fold reduction in measuring time. The consumption of costly deuterated solvent is reduced by at least 2 orders of magnitude. The 1-mm TXI microliter probe with z-gradient allows the measurement of one-dimensional 1H NMR and two-dimensional heteronuclear NMR spectra with a few nanomoles (micrograms) of compound with high sensitivity, speed, and quality. This is a breakthrough for discrete sample NMR spectroscopy with paramount importance for structure elucidation in natural compound chemistry and metabolic research. It offers also advantages for linking chromatographic methods to NMR in a nindustrial environment. Capillary tube NMR may find new applications in areas where high sample throughput is essential, e.g., in the quality control of large sample arrays from parallel chemistry, screening, and compound depositories. It has the potential to increase the sample throughput by 1 order of magnitude or more if new hardware for fast sample handling and exchange becomes available.     

Development of ultrahigh-throughput NMR spectroscopic analysis utilizing capillary flow NMR technology

An ultrahigh-throughput method for acquiring 1H NMR spectra is described. By constructing a continuous flow system utilizing an HPLC pump, autosampler, and a capillary flow NMR probe, it was possible to inject samples into the NMR spectrometer every 30 s using a continuous flow rate of 30 microL/min. 1H NMR spectroscopic data were acquired continuously into a pseudo-2D data file, with a 96-well-plate completed in <50 min. Spectra in continuous flow mode were readily obtained from approximately 3.4 mug (500 MHz), while the LOD was <850 ng. There was found to be little variation in either sample broadening within the flow system or signal intensities between multiple injections. This system offers several advantages over more conventional NMR spectroscopic analyses, notably the limited solvent required, high sensitivity, high speed, and improved spectral quality as a result of reduced spectral "dead" regions resulting from residual solvent levels.     

High-throughput nuclear magnetic resonance analysis using a multiple coil flow probe

An automated method for high-throughput nuclear magnetic resonance (NMR) spectroscopy has been developed using a four-coil Multiplex NMR probe. The probe is constructed with solenoidal microcoils optimized for detection of small volume, mass-limited samples and a flow-through design. Four samples can be simultaneously injected into the Multiplex probe with a robotics liquid handler and then analyzed in rapid succession using a selective excitation experiment. Due to the simultaneous injection of four samples and the reduced analysis time with rapid selective excitation, the analysis rate achieved thus far is as low as 1 sample/34 s for 1D 1H NMR.     

Capillary HPLC-NMR Coupling: High-Resolution (1)H NMR Spectroscopy in the Nanoliter Scale

Coupling HPLC and NMR is one of the most powerful techniques for simultaneous separation and structural elucidation of unknown compounds in mixtures. To date, however, minimizing the detection volume, as is required when coupling NMR with miniaturized separation techniques, has been accompanied by a dramatic loss in resolution of the NMR spectra. Here, we report on the coupling of gradient capillary HPLC with on-column, high-resolution NMR detection. On-line stopped-flow and static (1)H NMR spectra were acquired with capillary columns of 75-315 μm i.d. With detection over a length of 1.2 cm, cell volumes cover a range of 50-900 nL. An on-line-detected NMR separation of dansylated amino acids was carried out in a 315 μm i.d. fused silica capillary packed to a length of 12 cm with C(18) stationary phase. The low solvent consumption makes the use of fully deuterated solvents economically feasible. NMR spectra with resolution on the order of 3 Hz were obtained using a 50 nL detection cell to measure 1.1 nmol of dansylated γ-aminobutyric acid under static conditions in a 75 μm i.d. capillary.     

Cryogenic probe 13C NMR spectroscopy of urine for metabonomic studies

Cryogenic probe technology can significantly compensate for the inherently low sensitivity of natural abundance 13C NMR spectroscopy. This now permits its routine use in NMR spectroscopy of biofluids, such as urine or plasma, with acquisition times that enable a high throughput of samples. Metabonomic studies often generate numerous samples in order to characterize fully the time-dependent biochemical response to stimuli, but until now, they have been largely conducted using 1H NMR spectroscopy because of its high sensitivity and hence efficient data acquisition. Here, we demonstrate that information-rich 13C NMR spectra of rat urine can be obtained using appropriately short acquisition times suitable for biochemical samples when using a cryogenic probe. Furthermore, these data were amenable to automated pattern recognition analysis, which produced a profile of the metabolic response to the model hepatotoxin hydrazine that was consistent with earlier studies. Thus, a new source of detailed and complementary information is available to metabonomics using cryogenic probe 13C NMR spectroscopy.     

A microcoil NMR probe for coupling microscale HPLC with on-line NMR spectroscopy

An HPLC NMR system is presented that integrates a commercial microbore HPLC system using a 0.5-mm column with a 500-MHz proton NMR spectrometer using a custom NMR probe with an observe volume of 1.1 microL and a coil fill factor of 68%. Careful attention to capillary connections and NMR flow cell design allows on-line NMR detection with no significant loss in separation efficiency when compared with a UV chromatogram. HPLC NMR is performed on mixtures of amino acids and small peptides with analyte injection amounts as small as 750 ng; the separations are accomplished in less than 10 min and individual NMR spectra are acquired with 12 s time resolution. Stopped-flow NMR is achieved by diversion of the chromatographic flow after observation of the beginning of the analyte band within the NMR flow cell. Isolation of the compound of interest within the NMR detection cell allows multidimensional experiments to be performed. A stopped-flow COSY spectrum of the peptide Phe-Ala is acquired in 3.5 h with an injected amount of 5 micrograms.     

Chemical shift correlations from hyperpolarized NMR by off-resonance decoupling

Nuclear magnetic resonance, through observation of chemical shift, allows the separate identification of each atom in a molecule. Thus, NMR spectra impart an often unrivaled wealth of information on molecular structure. A particular advantage of NMR spectroscopy is the ability to record multidimensional spectra, which provide correlations between atoms. When compared to other techniques, such as optical spectroscopy, the acquisition of NMR spectra is however an insensitive process, requiring samples of high concentration and long acquisition times. Recently, it has been demonstrated that dynamic nuclear polarization, a hyperpolarization technique, can increase the NMR signal by several orders of magnitude. Here, we present a robust method that allows recording two-dimensional chemical shift correlations from such hyperpolarized molecules. The method makes use of an apparent scaling of the scalar coupling observed on one type of atom, when an off-resonance decoupling field is applied to another type of atom. Thus, two-dimensional chemical shift correlations can be read directly from a small number of scans acquired using a hyperpolarized sample. Due to the ease of implementing this technique on commercial hyperpolarization and NMR equipment, it appears ideally suited for routine application, for example, to obtain carbon-proton chemical shift correlations in organic molecules.     

Advancing NMR sensitivity for LC-NMR-MS using a cryoflow probe: application to the analysis of acetaminophen metabolites in urine

Cryogenic cooling of the NMR radio frequency coils and electronics to give greatly enhanced sensitivity is arguably the most significant recent advance in NMR spectroscopy. Here we report the first cryogenic probe built in flow configuration and demonstrate the application to LC-NMR-MS studies. This probe provides superior sensitivity over conventional noncryogenic flow NMR probes, allowing the use of 100 microL of untreated urine (40% less material than previous studies that required preconcentration) and yet revealing drug metabolites hitherto undetected by LC-NMR-MS at 500 MHz. Besides the known sulfate and glucuronide metabolites, previously undetected metabolites of acetaminophen were directly observable in a 15-min on-flow experiment. Simultaneous MS data also provided knowledge on the NMR-silent functional moieties. Further, stop-flow LC-NMR-MS experiments were conducted for greater signal-to-noise ratios on minor metabolites. The cryoflow probe enables the NMR analysis of lower concentrations of metabolites than was previously possible for untreated biofluids. This strategy is generally applicable for samples containing mass-limited analytes, such as those from drug metabolism studies, biomarker and toxicity profiling, impurity analysis, and natural product analysis.     

In situ electrochemical-NMR spectroscopy. Reduction of aromatic halides

A three-electrode, two-compartment cylindrical electrochemical cell has been constructed for in situ operation in a 500 MHz 1H NMR spectrometer. The cell was designed around a standard 10-mm sample tube holder and is lowered into position within the magnet by height-adjustable aluminum rods that support the wires connecting the potentiostat to the working, auxiliary and reference electrodes. The electrochemical cell functions under diffusion-controlled conditions in nonspinning mode and is able to operate under variable temperature and oxygen-free regimes. The working electrode consists of a 10-nm-thick gold film deposited onto the surface of a 7.49-mm-o.d. glass NMR tube that is located directly within the radio frequency coils of the NMR probe. The NMR spectra were collected simultaneously to the current flowing during the electrolysis with typical NMR line widths being approximately 2 Hz. The highly symmetrical nature of the cell design meant that probe tuning and gradient shimming changed minimally between experiments, which allowed standard shim sets to be saved and recalled for later use, thereby greatly reducing the time taken for tuning processes. Insertion and removal of the cell from within the magnet is achieved within a few seconds, which combined with the rapid shimming process makes the cell suitable for routine operation. The cell was used to study the in situ reduction of 9-bromoanthracene, 9-chloroanthracene, and 4-bromobenzophenone in deuterated acetonitrile with tetrabutylammonium hexafluorophosphate as the supporting electrolyte.     

Automated microflow NMR: routine analysis of five-microliter samples

A microflow CapNMR probe double-tuned for 1H and 13C was installed on a 400-MHz NMR spectrometer and interfaced to an automated liquid handler. Individual samples dissolved in DMSO-d6 are submitted for NMR analysis in vials containing as little as 10 microL of sample. Sets of samples are submitted in a low-volume 384-well plate. Of the 10 microL of sample per well, as with vials, 5 microL is injected into the microflow NMR probe for analysis. For quality control of chemical libraries, 1D NMR spectra are acquired under full automation from 384-well plates on as many as 130 compounds within 24 h using 128 scans per spectrum and a sample-to-sample cycle time of approximately 11 min. Because of the low volume requirements and high mass sensitivity of the microflow NMR system, 30 nmol of a typical small molecule is sufficient to obtain high-quality, well-resolved, 1D proton or 2D COSY NMR spectra in approximately 6 or 20 min of data acquisition time per experiment, respectively. Implementation of pulse programs with automated solvent peak identification and suppression allow for reliable data collection, even for samples submitted in fully protonated DMSO. The automated microflow NMR system is controlled and monitored using web-based software.     

Direct On-Line Coupling of Capillary HPLC with (1)H NMR Spectroscopy for the Structural Determination of Retinyl Acetate Dimers: 2D NMR Spectroscopy in the Nanoliter Scale

This paper presents the application of directly coupled capillary high-performance liquid chromatography (capillary HPLC) and proton high-field nuclear magnetic resonance spectroscopy (NMR) for structural elucidation of a so-far unknown kitol isomer. One- and two-dimensional continuous- and stopped-flow NMR spectra were recorded in a 180 μm i.d. capillary, corresponding to a detection volume of only 200 nL. Unequivocal structural assignment on the basis of 1D and 2D stopped-flow capillary HPLC-NMR experiments was performed. The kitol isomer mixture was present in a sample of thermally isomerized retinyl acetate and separated on a capillary column.     

MALDI quadrupole time-of-flight mass spectrometry: a powerful tool for proteomic research

A MALDI QqTOF mass spectrometer has been used to identify proteins separated by one-dimensional or two-dimensional gel electrophoresis at the femtomole level. The high mass resolution and the high mass accuracy of this instrument in both MS and MS/MS modes allow identification of a protein either by peptide mass fingerprinting of the protein digest or from tandem mass spectra acquired by collision-induced dissociation of individual peptide precursors. A peptide mass map of the digest and tandem mass spectra of multiple peptide precursor ions can be acquired from the same sample in the course of a single experiment. Database searching and acquisition of MS and MS/MS spectra can be combined in an interactive fashion, increasing the information value of the analytical data. The approach has demonstrated its usefulness in the comprehensive characterization of protein in-gel digests, in the dissection of complex protein mixtures, and in sequencing of a low molecular weight integral membrane protein. Proteins can be identified in all types of sequence databases, including an EST database. Thus, MALDI QqTOF mass spectrometry promises to have remarkable potential for advancing proteomic research.     

Application of a microcoil probe head in NMR analysis of chemicals related to the chemical weapons convention

A 1.7-mm microcoil probe head was tested in the analysis of organophosphorus compounds related to the Chemical Weapons Convention. The microcoil probe head demonstrated a high mass sensitivity in the detection of traces of organophosphorus compounds in samples. Methylphosphonic acid, the common secondary degradation product of sarin, soman, and VX, was detected at level 50 ng (0.52 nmol) from a 30-microL water sample using proton-observed experiments. Direct phosphorus observation of methylphosphonic acid with (31)P{(1)H} NMR experiment was feasible at the 400-ng (4.17 nmol) level. Application of the microcoil probe head in the spiked sample analysis was studied with a test water sample containing 2-10 microg/mL of three organophosphorus compounds. High-quality (1)H NMR, (31)P{(1)H} NMR, 2D (1)H-(31)P fast-HMQC, and 2D TOCSY spectra were obtained in 3 h from the concentrated 1.7-mm NMR sample prepared from 1 mL of the water solution. Furthermore, a 2D (1)H-(13)C fast-HMQC spectrum with sufficient quality was possible to measure in 5 h. The microcoil probe head demonstrated a considerable sensitivity improvement and reduction of measurement times for the NMR spectroscopy in identification of chemicals related to the Chemical Weapons Convention.     

Ratio analysis nuclear magnetic resonance spectroscopy for selective metabolite identification in complex samples

Metabolite identification in the complex NMR spectra of biological samples is a challenging task due to significant spectral overlap and limited signal-to-noise. In this study we present a new approach, RANSY (ratio analysis NMR spectroscopy), which identifies all the peaks of a specific metabolite on the basis of the ratios of peak heights or integrals. We show that the spectrum for an individual metabolite can be generated by exploiting the fact that the peak ratios for any metabolite in the NMR spectrum are fixed and proportional to the relative numbers of magnetically distinct protons. When the peak ratios are divided by their coefficients of variation derived from a set of NMR spectra, the generation of an individual metabolite spectrum is enabled. We first tested the performance of this approach using one-dimensional (1D) and two-dimensional (2D) NMR data of mixtures of synthetic analogues of common body fluid metabolites. Subsequently, the method was applied to (1)H NMR spectra of blood serum samples to demonstrate the selective identification of a number of metabolites. The RANSY approach, which does not need any additional NMR experiments for spectral simplification, is easy to perform and has the potential to aid in the identification of unknown metabolites using 1D or 2D NMR spectra in virtually any complex biological mixture.