Stopped-flow NMR

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. 

Fig. 2.3 Arrhenius ( ) and Eyring ( ) plots of data for the exchange of a single dmso molecule with Ga(dmso) + in CD3NO2. Nmr line-broadening was used for the higher temperatures and stopped-flow nmr experiments for the lower temperatures. For the Arrhenius plot, the slope and intercept (at T = 0) are -9.2 x 10 and +31,4 respectively, leading to = 76 kJ mol" and logA = 13.6. For the Eyring plot, the slope and intercept are —8.77 x 10 and +24.4 respeetively, leading to A// = 73 kJ mol" and AS = 5 J K" mol" .

Fig. 3.12 Parts of successively recorded H nmr spectra obtained by stopped-flow nmr after mixing Fe(dmso)g with [ H Jdmso in CDjNOj solution at 243.4 K. Fe(III) = 10 mM [ HjJdmso = 1.0 M. Resonances a = dmso b = SiMe4. Reproduced with permission from C. H. McAteer, P. Moore, J. Chem. Soc. Dalton Trans. 353 (1983).

The main prerequisite for on-line LC-NMR, besides the NMR and HPLC instrumentation, are the continuous-flow probe and a valve installed before the probe for the registration of either continuous-flow or stopped-flow NMR spectra. 

Since asterosaponin mixtures consist of a variety of compounds with different combinations of reoccurring structural features (oligosaccharide or steroidal side-chains), this approach has already yielded valuable information for identif-ing known constituents and for selecting compounds of interest for further investigations, such as 2D stop-flow NMR spectra (see Section 5.1.2.5 below). 

Figure 6.5 Stop-flow NMR spectra of selected peaks in fraction A. Reprinted with permission from Preiss, A., Sanger, U., Karfich, N., Levsen, K. and Miigge, C., Anal. Chem., 72, 992-998 (2000). Copyright (2000) American Chemical Society...

Figure 6.10 Stop-flow NMR spectra of the very early-eluting compounds of the leachate sample the chromatographic conditions are given on the figure. Spectrometer, Bruker DRX 600 probe head, 4 mm z-gradient LC probe between 256 and 1024 scans were acquired...

Figure 8.2.20 Schematic of the potential development of parallel CHPLC/NMR hyphenation systems. CHPLC is used for high separation efficiency, with a number of separations carried out simultaneously. Each separation is monitored either by using on-flow or stop-flow NMR detection using an RF microcoil, with each coil feeding data to a separate receiver...

It is a serious limitation that all these optical techniques can only be used for reactions that are accompanied by significant chromophoric changes, ft is an additional limitation of all these techniques that optical spectra tend to be broad and often show considerable overlap. Therefore reactive species can often not be monitored individually, even in cases where all of them have distinct spectral properties. Optical methods also provide little structural information about the various species in the mixture. The use of traditional stopped-flow is dependent upon the availability of optical signals to monitor change in fluorescence, absorbance or CD that occur upon binding or chemical conversion of substrate to product. In order to overcome these limitations of traditional stopped-flow optical techniques, various detection methods have been developed, such as stopped-flow mass spectrometry (MS), stopped-flow NMR, and stopped-flow EPR. 

The plant lectin concanavalin A, a metalloprotein isolated from the jack bean, has also received considerable attention. (588-590,630,631) Using the stopped flow NMR technique three distinct conformation states of the protein, when it is bound to Mn, Ca, and a-methyl-D-mannoside, (630) have been deduced. 

Various tools have been developed to enhance the potential of the techniqne, snch as mass-directed peak selection [55], on-line SPE for on-line preconcentration [56] or post-column peak trapping and stop-flow NMR [57]. 

There are a few ways of linking the techniques of LC-MS and LC-NMR. The most common method is in a parallel mode by splitting the flow, e.g. 50 1, so as to direct the majority of it to the NMR due to its relative insensitivity. This means that the analytes are detected simultaneously by both detectors and possibly also by UV, which may actually be used as the trigger to begin detection by the NMR and MS modules. Alternatively, the rapidly acquired MS data can be used to direct the NMR experiments or vice versa. A second method of interfacing the two techniques is to use the serial mode or stopped flow mode, which enables more sensitive NMR experiments to be carried out. A recent development in stopped flow NMR is the inclusion of in-line solid phase extraction (SPE) after the LC. The SPE acts as a fraction collector for individual compounds. This trapping/ washing step can improve sensitivity several fold. A third method is fraction collection, where samples from the LC are collected in a loop for analysis later, perhaps after certain data have been reviewed. 

S/N for a flow cell of only 50 nL volume. The main drawback was rather wide NMR linewidths. Behnke etal. demonstrated capillary LC/NMR in 1996 [138], using flow cells of 50 nL and 900nL and a standard saddle coil, and achieved sensitivities approximately 10 times greater than conventional LC/NMR. The approach, however, did not incorporate a UV detection cell so that stopped-flow NMR measurements had to be nuule by reference to on-flow NMR measurements. 

Two essential carotenoids, lutein and zeaxanthin, play an important role in the visual process. For the treatment of age-related macular degeneration (AMD), knowledge of the isomeric composition within the macula is of particular interest [7], Full assignment of isomeric configuration is possible by the registration of two-dimensional proton-proton correlated NMR spectra. Figure 7-7 shows, as an example, the COSY stopped-flow NMR spectrum of all-trans zeaxanthin isolated from ox retina. 

The on-line stop-flow NMR spectra were acquired on a Varian UNITY-INOVA 600 MHz NMR spectrometer with a 60 pi triple resonance... 

At the other kinetic extreme, reactions with A < 10 may be investigated by flow or stopped-flow NMR (A <1). In this manner one may extend the temperature range of Arrhenius plots so as to reduce their all too prevalent errors. Observed line shapes in flow FT NMR can become complex. 

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