Stopped flow analyzer

Online detection using 4H nuclear magnetic resonance (NMR) is a detection mode that has become increasingly practical. In a recent application, cell culture supernatant was monitored on-line with 1-dimensional NMR for trehalose, P-D-pyranose, P-D-furanose, succinate, acetate and uridine.33 In stopped-flow mode, column fractions can also be analyzed by 2-D NMR. Reaction products of the preparation of the neuromuscular blocking compound atracurium besylate were separated on chiral HPLC and detected by 4H NMR.34 Ten isomeric peaks were separated on a cellulose-based phase and identified by online NMR in stopped-flow mode. 

In order to use the stopped-flow technique, the reaction under study must have a convenient absorbance or fluorescence that can be measured spectrophotometri-cally. Another method, called rapid quench or quench-flow, operates for enzymatic systems having no component (reactant or product) that can be spectrally monitored in real time. The quench-flow is a very finely tuned, computer-controlled machine that is designed to mix enzyme and reactants very rapidly to start the enzymatic reaction, and then quench it after a defined time. The time course of the reaction can then be analyzed by electrophoretic methods. The reaction time currently ranges from about 5 ms to several seconds. 

The dynamics of intercalation of small molecules with DNA, groove binding and binding to specific sites, such as base pair mismatches have been studied by stopped-flow,23,80 108 temperature jump experiments,26,27,94 109 120 surface plasmon resonance,121 129 NMR,86,130 135 flash photolysis,136 138 and fluorescence correlation spectroscopy.64 The application of the various techniques to study the binding dynamics of small molecules will be analyzed for specific examples of each type of binding. 

LC-NMR can be used to identify natural products in crnde plant extracts that usually consist of complex mixtnres. The crnde natural product extracts normally contain a great nnmber of closely related and difficult-to-separate compounds. The classical separation approach may become very tedious and time-consuming. The directly conpled HPLC-NMR presents an efficient separation techniqne together with a powerfnl spectroscopic method to speed up the identification process. LC-NMR has been nsed extensively for characterization of natnral prodncts. More recently, the combination of LC-NMR and LC-MS has been further developed in this field. Eor example, Wilson et al. have nsed combined on-flow NMR and electrospray ionization MS to characterize ecdysteroids in extracts of silene otites. After reversed-phase HPLC nsing D2O in acetonitrile-dj and UV detection, the LC flow was split 95 5 for the simnl-taneous detection by NMR and MS. The peaks of interest were analyzed by stop-flow NMR to give better quality spectra for structural assignment. 

A derivative technique, known as stopped-flow/tempera-ture-jump, allows one to take advantage of the stopped-flow strategy to establish a steady-state rate condition which can then be perturbed and analyzed by using the elements of a temperature-jump device. 

Johnson and Fierke Hammes have presented detailed accounts of how rapid reaction techniques allow one to analyze enzymic catalysis in terms of pre-steady-state events, single-turnover kinetics, substrate channeling, internal equilibria, and kinetic partitioning. See Chemical Kinetics Stopped-Flow Techniques... 

Comprehensive 2D HPLC can be also operated under stop-flow mode. In this case, after transferring a desired fraction volume onto the secondary column, the flow of the mobile phase in the first dimension is stopped and the fraction analyzed in the second dimension. When the separation is finished, the mobile-phase flow in the first dimension is switched on and the whole procedure is repeated again for the analysis of all the transferred fractions. The disadvantage of this procedure is the long analysis time, while the advantage can be that the second-dimension column can give higher plate numbers if compared to the continuous approach [23]. 

Several studies have dealt with the influence of lipids on conformational equilibria in cytochrome c via hydrophobic and electrostatic interactions. The binding of sodium dodecyl sulfate monomers and micelles was reported to cause a transition of cytochrome c to a state B2 which is of potential physiological relevance. The interplay between heme only state changes and secondary structure changes was analyzed by freeze-quench and stopped-flow experiments.276 The response of the heme spin state to lipid acyl chains in cytochrome c was... 

The interaction of SelB with its ligands was analyzed by stopped flow methods. The main finding was that binding of the two RNA ligands (selenocysteyl-tRNA and the SECIS element) confers to the SelB protein, a conformation that... 

The capability of analyzing a complex mixture in a chromatographic run by the hyphenation of several techniques, such as NMR and MS, to HPLC is becoming more popular in the pharmaceutical industry. NMR and MS data on the same analyte are crucial for structural elucidation. When different isolates such as metabolites are analyzed by NMR and MS, one cannot always be certain that the NMR and the MS data apply to the same analyte, especially when the analytes have been isolated using analytical columns and prep columns for the MS and NMR analysis, respectively. HPLC conditions are not always reproducible when analytical and prep-HPLC columns are used to isolate different amounts of the analytes of interest. To avoid this ambiguity, LC-MS and LC-NMR are combined. MS data should be obtained initially because with NMR, data collection in the stop-flow mode can take hours or days, depending on the complexity of the structure and the amount of sample. This is why it is preferable to designate this operation as LC-MS-NMR rather than LC-NMR-MS or LC-NMR/MS. 

As mentioned in the section of modes of operation for LC-NMR (Section 20.3.2), with the use of shielded cryomagnets, the location of the MS instrument will follow the same rule as for the HPLC. The most common modes of operation for LC-MS-NMR are on-flow and stop-flow. With stop-flow, the MS instrument can also be used to stop the flow on the chromatographic peak of interest that is to be analyzed by NMR. These two modes are presented here with an example. In the loop collection mode, the MS of the LC-MS-NMR system may also monitor the trapping of the chromatographic peak inside the loop. 

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