High-sensitivity stop-flow

Both MS and NMR coupling to HPLC have been employed for the analysis of p-carotene isomers and determination of lutein and zeaxanthin isomers in spinach, sweet com, and in retina. Capillary high performance hquid chromatography with stop flow connected to NMR (600 MHz) was used for stracture elucidation of all-trans deoxylutein 11 and its isomers.Efforts are in progress to eliminate the remaining major drawbacks such as obligatory use of deuterated solvents in the mobile phase, poor sensitivity, and low throughput of HPLC-NMR analyses. 

The coupling of HPLC with NMR represents a powerful method for the high-throughput screening of peptides in mixtures run in stop-flow and continuous-flow modes. It is possible to obtain routine high-quality HPLC/NMR ID NMR data with as little as 5 pg of compound in a chromatogram peak. However, the HPLC/NMR technique cannot be favorably compared to mass spectrometry techniques (HPLC/MS) in terms of sensitivity and speed of analysis. To date, the majority of reports of the use of HPLC/NMR have been for drug metabolites.1 ... 

Watanabe and Niki introduced the coupling of NMR to LC as an on-line detector [20]. After these initial stopped-flow experiments, Bayer et al. [21] reported the first continuous-flow LC-NMR experiment. However, a number of impediments associated with LC-NMR hindered routine analytical application for a number of years. Since then, new instrumentation and analytical methodologies for LC-NMR have been developed and commercialized. The development of high-field-strength magnets, better solvent-suppression techniques, more sensitive small-diameter transmitter/receiver coils, on-column sample preconcentration, and expanded flow cells have improved the sensitivity of LC-NMR. 

The use of Ty fluorescence quenching as a probe provides a direct spectroscopic probe of fluoride binding to Tymet. Stopped-flow fluorimetric experiments can be performed with high sensitivity and speed, thereby avoiding the need for large amounts of protein and the experimental difficulties arising from the intrinsic instability of the enzyme, the latter being important especially at the lower pH values. 

The HCyDTA produced in turn reacts rapidly with lead ions, and there is no direct replacement of barium ions by lead ions. The rate of the reaction therefore can be adjusted by pH control. Some of the reactions are so fast that a pH as high as 8 is required for stopped-flow techniques to be applicable at the same time, this high pH makes copper and cobalt (instead of lead) unsuitable as scavenging metals. The sensitivity is such that as little as 10 M metal ion can be measured. It is important... 

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