Stopped-flow approach

Stopped-flow approaches have interesting features for CL monitoring in very fast CL reactions. In this case, the sample and reagent are efficiently mixed in a reaction chamber and rapidly forced to an observation cell, in which the flow is violently stopped, allowing monitoring of the full intensity-time curve and allowing kinetics measurements (Fig. 7), which can be related to the analyte... 

A stopped flow approach can be employed using dialysis sampling which allows effective transport of low molecular weight components from the sample across the dialysis membrane and hence the dialysate will accurately reflect the composition of the sample. 

Having shown that the HPLC-NMR system could separate and characterise these three substances by using both continuous-flow and stop-flow approaches, a more realistic test was applied. This successfully characterised the two components shown below with the impurity at a level of 3 % using stop-flow HPLC-NMR ... 

The second example is the measurement of the kinetics of a substrate binding to extracted FeMo-cofactor of nitrogenase by a double-mixing stopped-flow approach. In a typical experiment, solutions of FeMoco-L and CN are rapidly mixed and held together for a known length of time. Subsequently, this solution is mixed with a solution of [NEt4]SPh whereupon the thiolate reacts with the cofactor. 

The direct coupling of HPLC with NMR spectroscopy has waited for a number of technological developments to make it a feasible routine technique. Since then, major advances have made the routine use of on-line NMR detection of HPLC fractions a useful adjunct to the armoury of analytical methods. " Experiments can be carried out in one of three modes, direct on-line NMR detection of the HPLC eluent (on-flow), a stopped-flow approach or finally the eluted fractions can be stored in capillary tubes for later recall for detailed NMR spectroscopic studies. No compromise needs to be made in the chromatographic conditions, and programmed gradient elution profiles can be accommodated. The extension of the method to nanolitre scale separations has also been discussed. ... 

Karakosta, T. D., P. D. Tzanavaras, and D. G. Themelis. 2011. Automated pre-column derivatization of thiolic fruit-antibrowning agents by sequential injection coupled to high-performance liquid chromatography using a monolithic stationary phase and an in-loop stopped-flow approach. J. Sep. Sci. 34 2240-2246. 

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