Steep solvent gradient NMR analysis method

Details Steep solvent gradient NMR analysis method Steep solvent gradient NMR analysis method

2001-12-03

2004-03-02

Arana, Louis (Department: 2859)

Electricity: measuring and testing

Particle precession resonance

Spectrometer components

C324S306000

Reexamination Certificate

active

06700379

ABSTRACT:

TECHNICAL FIELD
This invention relates to methods and systems for NMR analysis of analytes in small volumes. More particularly, this invention relates to methods and systems employing NMR microcoils for analysis of analytes in, for example, nanoliter sample volumes.
BACKGROUND
Nuclear magnetic resonance spectroscopy, or NMR, is a powerful and commonly used method for analysis of the chemical structure of molecules. NMR provides spectral information as a function of the electronic environment of the molecule and is nondestructive to the sample. In addition, reaction rates, coupling constants, bond-lengths, and two- and three-dimensional structure can be obtained with this technique. NMR combined with liquid chromatography or capillary electrophoresis was demonstrated as early as 1978 using stopped flow (Watanabe et al. (1978) Proc. Jpn. Acad. 54:194), and in 1979 with continuous flow (Bayer et al. (1979) J. Chromatog. 186:497-507), though limitations due to solvent as well as inherent sensitivity curtailed the use of the method. That is, the inherent insensitivity of the NMR method limited its usefulness as a detection method for liquid phase analysis of very small samples, such as effluent from an analyte focusing or concentrating method (those two terms being used here interchangeably unless otherwise indicated), e.g., a liquid chromatography or capillary electrophoretic separation. Conventional NMR spectrometers typically use relatively large RF coils (mm to cm size) and samples in the mu.l to ml volume range, and significant performance advantages are achieved using NMR microcoils when examining very small samples. NMR microcoils are known to those skilled in the art and are shown, for example, in U.S. Pat. No. 5,654,636 to Sweedler et al., and in U.S. Pat. No. 5,684,401 to Peck et al., and in U.S. Pat. No. 6,097,188 to Sweedler et al., all three of which patents are incorporated herein by reference in their entireties for all purposes. A solenoid microcoil detection cell formed from a fused silica capillary wrapped with copper wire has been used for static measurements of sucrose, arginine and other simple compounds. Wu et al. (1994a), J. Am. Chem. Soc. 116:7929-7930; Olson et al. (1995), Science 270:1967-1970, Peck (1995) J. Magn. Reson. 108(B) 114-124. Coil diameter has been further reduced by the use of conventional micro-electronic techniques in which planar gold or aluminum R.F. coils having a diameter ranging from 10-200 .mu.m were etched in silicon dioxide using standard photolithography. Peck 1994 IEEE Trans Biomed Eng 41(7) 706-709, Stocker 1997 IEEE Trans Biomed Eng 44(11) 1122-1127, Magin 1997 IEEE Spectrum 34 51-61, which are also incorporated herein by reference in its entirety for all purposes.
Miniature total analysis systems (&mgr;-TAS) are discussed in Integrating Microfluidic Systems And NMR Spectroscopy—Preliminary Results, Trumbull et al, Solid-State Sensor and Actuator Workshop, pp. 101-05 (1998), Magin 1997 IEEE Spectrum 34 51-61, and Trumbull 2000 47(1) 1-6 incorporated herein by reference in its entirety for all purposes. The Trumbull et al. device integrated multiple chemical processing steps and the means of analyzing their results on the same miniaturized system. Specifically, Trumbull et al. coupled chip-based capillary electrophoresis (CE) with nuclear magnetic resonance spectroscopy (NMR) in a &mgr;L-TAS system. Linewidths of 1.4 Hz have been demonstrated using single turn planar NMR coils integrated with microfluidic channels.
Capillary-based liquid chromatography and microcoil NMR have compatible flow rates and sample volume requirements. Thus, for example, the combination of the Waters CapLC™ available from Waters Corporation (Milford, Mass., USA) and the MRM CapNMR™ flow probe available from MRM Corporation (Savoy, Ill.), a division of Protasis Corporation (Marlborough, Mass., USA) provides excellent separation capability in addition to UV-VIS and NMR detection for mass-limited samples. The Waters CapLC™ has published flow rates from 0.02 &mgr;L/minute to 40 &mgr;L/minute. A typical CapLC on-column flow rate is 5 &mgr;&mgr;L/min, the autosampler-injected analyte volume is 0.1 &mgr;L or more, and accurate flow rates are achieved through capillary of typically 50 &mgr;m inner diameter. The NMR flow cell has a typical total volume of 5 &mgr;L with a microcoil observe volume of 1 &mgr;L. A typical injected sample amount for CapLC-&mgr;NMR analysis is a few &mgr;g (nmol) or less.
Conventional scale LC-NMR systems (flow rates about 1 ML/min) typically employ solvent gradients of 2% per minute to 4% per minute. The probes have typical flow cell volumes of about 150 &mgr;L and typical observe volumes of 30-60 &mgr;L. In such systems, the application of a solvent gradient followed by stopped flow can initially result in severely distorted NMR line shapes due to the magnetic inhomogeneity caused by nonuniform solvent composition in the flow cell. After a reasonable equilibration time, if the solvent gradient is no longer too steep, line shape is seen to recover from the initial stopped flow conditions. However, steep solvent gradients typically result inmagnetic distortions so severe that NMR is practically precluded due to the extremely long recovery time (>>1 hr) necessary to achieve high spectral resolution.
Capillary scale systems also are known. In such systems, a capillary-based analyte extraction chamber can be connected to an NMR flow cell, such as by being positioned as an operation site along a capillary channel extending to the NMR flow cell. Exemplary such integrated capillary-based analyte extraction chambers are shown in U.S. Pat. No. 6,194,900, the entire disclosure of which is incorporated herein by reference for all purposes.
There is a need in the art for improved NMR methods for analyzing analytes. There is a particular need for improved NMR methods of analyzing small samples, especially samples of less than 10 &mgr;L or even less than 1.0 &mgr;L.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.
SUMMARY
Solvent gradients play an important role in NMR analysis, as indicated above. In accordance with the methods disclosed here, a flow of analyte sample fed to an NMR flow cell has a mobile phase with advantageously steep solvent gradient.
In accordance with a first aspect, a method of analyzing an analyte comprises feeding a fluid flow of analyte sample fluid, that is a fluid containing or suspected of possibly containing one or more analytes of interest in a multi-component mobile phase to a fluid channel of a nuclear magnetic resonance (NMR) probe. The analyte sample fluid is fed to an NMR flow cell in the fluid channel. The flow cell comprises an RF microcoil operably associated with an enlarged containment region. The mobile phase of the analyte sample flowing to the NMR flow cell has a solvent gradient greater than 10% per minute. In certain preferred embodiments, the mobile phase of the analyte sample fluid has a solvent gradient of 10%-30% per minute or more. Coupled with NMR detection, this variation in (mobile phase) solvent composition is fed directly into the NMR flow cell. Exemplary mobile phases include aqueous/organic mobile phases, and any others suitable to the particular application, of which many are known to those skilled in the art. It will be recognized that the methods and systems disclosed here provide an advantageous technological advance. An operator of a liquid chromatography system or other analyte extraction means generally wants full access and control over the solvent composition of the analyte sample flow, as this chemistry directly influences the effectiveness of the separation. In many instances, the compositional mixture of two or more solvents will be intentionally varied to provide a desired effect. The necessary or useful rate of solvent compositional change is in some ins

Affiliated with

Also associated with

Rating

Steep solvent gradient NMR analysis method does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Steep solvent gradient NMR analysis method, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Steep solvent gradient NMR analysis method will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3280999