Direct Stop-Flow

It is also possible to store fractions of the chromatogram intermediately in sample loops. From these storage loops the samples are transferred into the NMR detection cell, after when the separation has finished. The NMR measurement is then again carried out under static conditions. We refer to the combination of these two procedures as loop storage/loop transfer. 

Measurement under static conditions Direct stop-flow 

the outlet of the chromatographic system is connected to an NMR detection cell. The NMR spectra are acquired continuously while the sample is flowing through the detection cell. The result is a set of one-dimensional (ID) NMR spectra which cover the whole chromatogram and are typically displayed as a two-dimensional (2D) matrix showing NMR spectrum against retention time, similar to an LC-diode array detection (DAD) plot. 

The chromatography and NMR systems perform completely independent by of each other. The only necessary link is the liquid connection between the column and the NMR detection cell. The NMR spectrometer can act as a detector for the chromatographic system, so that even a conventional LC detector in the chromatographic system is not necessary. 

The typical peak width with analytical columns of 4.6 mm i.d. and a 1 ml/min flow rate is of the order of 10-30 s. The acquisition of NMR spectra with a short relaxation delay and an acquisition time of below 1 s allows the acquisition of 8-24 transients for one spectrum during the presence of a peak in the NMR cell. This low number of transients limits the detectable amount of sample to 5-10 xg per compound. 

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We have also shown that the indirect SOD assays, which are the mostly used methods for demonstrating complex SOD activity, are not very reliable and if, they can be applied only upon considering possible cross reactions between indicator substance and the studied complex in their different oxidation forms, in which they may occur within the SOD catal5rtic cycle. The direct stopped-flow method, where the high excess of superoxide over complex can be utilized, is a better probe for... 

There are four general modes of operation for LC-NMR on-flow, direct stop-flow, time-sliced and loop collection/transfer. The mode selected will depend on the level and complexity of the analyte and also on the NMR information required. All modes of LC-NMR can be run under full automation for LC peak-picking, LC peak transfer to storage loops or NMR flow cell, and NMR detection [46],... 

Figure 2.3 Peak broadening effects of the (a) direct stop-flow and (b) loop-storage/loop transfer procedures. The amounts of washing solvent required is defined by the chromatographic separation of the peaks in the direct stop-flow mode, while being user-defined in the loop transfer mode...

The chromatographic stage is not interrupted and therefore no stop-start effects will create disturbances. The peaks are separated in the storage loops, and therefore the NMR measurement time is not limited and will not decrease the performance of other peaks. In complex chromatograms the chance of finding the peak(s) of interest is dramatically increased. As in the direct stop-flow mode, the static conditions provide stability and the best NMR conditions for the acquisition of all kinds of high-resolution ID and 2D NMR spectra. 

The other methods of improving S/N and spectral resolution are to employ peak-directed stopped flow LC/NMR or loop-capture. The former is a... 

Hgure 2 Typical setup used for on-flow and direct stop-flow LC/NMR experiments. The control of the stop-flow valve is achieved by the computer of the LC/UV system which triggers both the valve of the HPLC pump and the NMR acquisition computer. A calibrated delay is used for parking the LC-peak of interest with precision at the center of the LC/NMR flow probe. 

Another way to improve the S/N ratio is the recording of the spectra in stop-flow mode. Operation in the direct stop-flow mode requires that the retention times of the analytes of interest are known or that a sensitive method of detection such as LC/UV or LC/ MS is used prior to LC/NMR to trigger the detection. In practice, one of these detectors is connected online before the NMR instrument and the signal of the analytes of interest passing through this detector is used to trigger a valve that will stop the LC flow exactly when the peak reaches the NMR cell after a calibrated delay (for setup, see Figure 2). 

The stop-flow mode can also be operated in a loop storage loop-loop transfer mode. In this case, the LC peaks are stored in a storage device consisting of multiple loops. The analytes are parked in these loops without interrupting the separation. At a later stage the content of the loop is transferred in the LC/ NMR flow cell like in the direct stop-flow mode. This process in two steps has the advantage that no disturbance due to the multiple stops in the separation is recorded and that no limitation due to diffusion effects occms furthermore, the transfer in the loops is completely independent from the NMR measurements and can be made separately. The mode of operation can be completely automated and different series of NMR experiments can be programmed in advance for each analyte of interest. 

Horizontal pipes should slope slightly downwards in the direction of flow, where this can be arranged. If a suction or discharge line has to rise, the size may be decreased to make the gas move faster. In the case of a lift of more than 5 m, a trap should be formed at the bottom to collect any oil which falls back when the plant stops [33]. 

Temperature jump method. " Stopped flow method. Direct spectrophotometry. 

Many transition metal complexes have been considered as synzymes for superoxide anion dismutation and activity as SOD mimics. The stability and toxicity of any metal complex intended for pharmaceutical application is of paramount concern, and the complex must also be determined to be truly catalytic for superoxide ion dismutation. Because the catalytic activity of SOD1, for instance, is essentially diffusion-controlled with rates of 2 x 1 () M 1 s 1, fast analytic techniques must be used to directly measure the decay of superoxide anion in testing complexes as SOD mimics. One needs to distinguish between the uncatalyzed stoichiometric decay of the superoxide anion (second-order kinetic behavior) and true catalytic SOD dismutation (first-order behavior with [O ] [synzyme] and many turnovers of SOD mimic catalytic behavior). Indirect detection methods such as those in which a steady-state concentration of superoxide anion is generated from a xanthine/xanthine oxidase system will not measure catalytic synzyme behavior but instead will evaluate the potential SOD mimic as a stoichiometric superoxide scavenger. Two methodologies, stopped-flow kinetic analysis and pulse radiolysis, are fast methods that will measure SOD mimic catalytic behavior. These methods are briefly described in reference 11 and in Section 3.7.2 of Chapter 3. 

Stopped-flow measurements with superoxide in aqueous solution at physiological pH are not possible due to its fast self-dismutation under these conditions. Therefore, the indirect assays such as McCord-Fridovich, adrenalin and nitroblue tetrazolium (NET) assays are widely used in the literature, not only for qualitative but also for quantitative detection of SOD activity of small molecular weight mimetics 52). Not going into details, we just want to stress that the indirect assays have very poor even qualitative reliability, since they can demonstrate the SOD activity of the complexes which does not react with superoxide at all. It has been reported in the literature that this is caused by the interference of hydrogen peroxide 29). We have observed that the direct reaction between complexes and indicator... 

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. 

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