HPLC with superheated water

A new topic is now included Chapter 20 about quahty assurance. Part of it could be found before in chapter 19 but now the subject is presented much broadly and independent of Analytical HPLC . Two chapters in the appendix were updated and expanded by Bruno E. Lendi, namely the ones about the instrument test (now chapter 25) and troubleshooting (now chapter 26). Some new sections were created 1.7, comparison of HPLC with capillary electrophoresis 2.11, how to obtain peak capacity 8.7, van Deemter curves and other coherences 11.3, hydrophilic interaction chromatography 17.2, method transfer 18.4, comprehensive two-dimensional HPLC 23.3, fast separations at 1000 bar 23.5, HPLC with superheated water. In addition, many details were improved and numerous references added. 

In reeent years, tire use of elevated temperatures has been reeognised as a potential variable in method development. Witlr inereased temperature, aqueous-organie mobile phases separations ean improve, viseosity deereases and diffusion inereases so baek pressures are redueed. At higher temperatures (usually with superheated water > 100 °C under modest pressures) water alone ean be used as the mobile phase and eair provide unique separation opportunities. The absenee of an organie solvent enables the use in HPLC of alternative deteetors sueh as FID or on-line LC-NMR using deuterium oxide as the eluent. 

B. A. Ingelse, H. G. Janssen, and C. A. Cramers, HPLC-FID with superheated water as the effluent Improved methods and instrumentation, J. High Resolut. Chromatogr. 21 (1998), 613-616. 

As alluded to earlier, even though the major problems associated with the need for suppression of the signals for the solvents used in HPLC-NMR have been addressed to the extent that the techniques can now be routinely employed, avoidance of the problem still remains an area of interest. In recent years, there has been a resurgence of interest in the use of hot and even superheated water... 

Most recently, a further study has been performed using superheated-water HPLC with NMR and MS to analyse a mixture of sulphonamides [68]. The chromatography was performed as before with D20-phosphate buffer (pD 3.0) as eluent. A temperature gradient from 160 to 200 °C at 2°C min-1 was employed. A mixture of four sulphonamides, i.e. sulacetamide, sulphadiazine, sulfamerazine and sulfamethazine, was separated in this system with UV, NMR and MS detection. It rapidly became clear from a study of the spectroscopic data that while sulfacetamide and sulfadiazine gave the expected NMR and mass spectra, those for sulfamerazine and sulfamethazine did not. These compounds gave spectra that were 3 and 6 mass units higher than expected,... 

These investigations of the use of superheated water with NMR spectroscopy are still at an early stage and have not yet been applied to real problems. Many questions remain to be answered concerning the suitability of this chromatography for thermally labile compounds, and with the current stationary phases available the technique is probably limited to moderately polar compounds. However, the technique is readily implemented and may in time prove to be a useful addition to the armoury of HPLC-NMR methods. 

As demonstrated from the 312 relevant SciFinder references, ecdysteroids have been isolated by RP-HPLC,176-179 HP-NPC,180-182 HP-IEX,183,184 or HP-AC.185 The dominant mode for the separation of ecdysteroids is HP-RPC, with numerous published applications including the use of superheated water as a mobile phase.186... 

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