Stopped-flow chromatography

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. 

Figure 7.21 Stopped-flow 111 NMR spectrum (400 MHz) of bis(2-ethylhexyl)phthalate from PVC with supercritical CO2. After Albert [152]. Reprinted from Journal of Chromatography, A785, K. Albert, 65-83, Copyright (1997), with permission from Elsevier...

There are several variations on the theme of instrument set up, which have been used in an attempt to overcome the shortcomings inherent in the concept. For example, as an alternative to the stop-flow method, the various fractions can be collected into sample loops (small loops of capillary tubing) which can then be flushed into the flow cell and studied at leisure. After spectroscopic examination, each sample can then be returned to its loop and the next pumped in. Fractions suffer dilution in this way but this approach would seem to offer an advantage over stop-flow in that at least the chromatography is not compromised by diffusion on the column. 

These experiments were carried out mainly by NMR spectrometry ( H and ljC) and high performance liquid chromatography. Stopped-flow and low temperature techniques were used in some of the NMR experiments. In all cases the reactions were aimed at monitoring the half-life of the starting materials as well as monitoring the products which were formed. Detailed procedures will appear shortly [15]. 

Optical Spectroscopy General principles and overview, 246, 13 absorption and circular dichroism spectroscopy of nucleic acid duplexes and triplexes, 246, 19 circular dichroism, 246, 34 bioinorganic spectroscopy, 246, 71 magnetic circular dichroism, 246, 110 low-temperature spectroscopy, 246, 131 rapid-scanning ultraviolet/visible spectroscopy applied in stopped-flow studies, 246, 168 transient absorption spectroscopy in the study of processes and dynamics in biology, 246, 201 hole burning spectroscopy and physics of proteins, 246, 226 ultraviolet/visible spectroelectrochemistry of redox proteins, 246, 701 diode array detection in liquid chromatography, 246, 749. 

Fischer CH, Weller H, Fojtik A, Lumepereira C, Janata E, Henglein A (1986) Photochemistry of Colloidal Semiconductors, 10. Exclusion Chromatography and Stop Flow Experiments on the Formation of Extremely Small Cds Particles. Ber Bimsen-ges 90 46-49... 

Frequently industrial hygiene analyses require the identification of unknown sample components. One of the most widely employed methods for this purpose is coupled gas chromatography/ mass spectrometry (GC/MS). With respect to interface with mass spectrometry, HPLC presently suffers a disadvantage in comparison to GC because instrumentation for routine application of HPLC/MS techniques is not available in many analytical chemistry laboratories (3). It is, however, anticipated that HPLC/MS systems will be more readily available in the future ( 5, 6, 1, 8). HPLC will then become an even more powerful analytical tool for use in occupational health chemistry. It is also important to note that conventional HPLC is presently adaptable to effective compound identification procedures other than direct mass spectrometry interface. These include relatively simple procedures for the recovery of sample components from column eluate as well as stop-flow techniques. Following recovery, a separated sample component may be subjected to, for example, direct probe mass spectrometry infra-red (IR), ultraviolet (UV), and visible spectrophotometry and fluorescence spectroscopy. The stopped flow technique may be used to obtain a fluorescence or a UV absorbance spectrum of a particular component as it elutes from the column. Such spectra can frequently be used to determine specific properties of the component for assistance in compound identification (9). 

The direct coupling of liquid chromatography with proton NMR has been attempted numerous times. Early experiments of coupled HPLC- H-NMR were conducted in a stop-flow mode or with very low flow rates [193-195]. This was necessary to accumulate a sufficient number of spectra per sample volume in order to improve the signal-to-noise ratio. Other problems associated with the implementation of on-line HPLC-NMR have included the need for deuterated solvents. However, with the exception of deuterium oxide, the use of deuterated eluents is too expensive for routine analysis. Therefore, proton-containing solvents, such as acetonitrile or methanol, must be used. To get rid of the solvent signals in the spectra, the proton NMR signals of the solvents have to be suppressed. 

Dead time In column chromatography, the time, r, required for an unretained species to traverse the column in stopped-flow kinetics, the time between the mixing of reactants and the arrival of the mixture at the observation cell. 

Stop-flow injection In high-performance liquid chromatography, introduction of the sample at the head of the column while solvent flow is temporarily discontinued. 

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