No abstract

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.

Time-resolved NMR spectroscopy is used to studychanges in protein conformation based on the elapsed time after a change in the solvent composition of a protein solution. The use of a micromixer and a continuous-flow method is described where the contents of two capillary flows are mixed rapidly, and then the NMR spectra of the combined flow are recorded at precise time points. The distance after mixing the two fluids and flow rates define the solvent-protein interaction time; this method allows the measurement of NMR spectra at precise mixing time points independent of spectral acquisition time. Integration of a micromixer and a microcoil NMR probe enables low-microliter volumes to be used without losing significant sensitivity in the NMR measurement. Ubiquitin, the model compound, changes its conformation from native to A-state at low pH and in 40% or higher methanol/water solvents. Proton NMR resonances of the His-68 and the Tyr-59 of ubiquitin are used to probe the conformational changes. Mixing ubiquitin and methanol solutions under low pH at microliter per minute flow rates yields both native and A-states. As the flow rate decreases, yielding longer reaction times, the population of the A-state increases. The micromixer-NMR system can probe reaction kinetics on a time scale of seconds.

Two designs for incorporating multiple solenoidal microcoils into a single probe head are presented to increase the throughput of high-resolution NMR. Through a combination of radio frequency switches and low-noise amplifiers, multiple NMR spectra can be acquired in the same time as a single spectrum from a conventional probe consisting of one coil. Since this method does not compromise sensitivity with regard to the individual microcoils, throughput increases linearly with the number of coils. Only one receiver is needed, and data acquisition parameters can be optimized for each sample. Specifically, a four-coil system has been implemented for proton NMR at 250 MHz using a wide-bore magnet, with an observe volume of 28 nL for each microcoil. Signal cross-contamination was approximately 0.2% between individual coils, and simultaneous one- and two-dimensional spectra have been obtained from samples of fructose, galactose, adenosine triphosphate, and chloroquine (7 nmol of each compound). A more compact two-coil configuration has also been designed for operation at 500 MHz, with observe volumes of 5 and 31 nL for the two coils. One- and two-dimensional spectra were acquired from samples of 1-butanol (55 nmol) and ethylbenzene (250 nmol).