Multistep HPLC

The reader is directed to Refs. 40, 42,43, 60, and 61 for selected practical approaches to multistep HPLC of peptides, a brief review of which can be found in Ref. 62. Proteomics research has led to an increasing demand for high-throughput separation of proteins and their identification. Such multistep and fully automated HPLC systems are in development [55,63]. The coupling of HPLC to electrospray ionization (ESI) mass spectrometry for the identification of peptides and proteins is revolutionizing proteomic research. However, we are focusing on the basic understanding of HPLC in this chapter. 

AEOs have been analysed by HPLC and UV or fluorescence detection after suitable derivatisation. The derivatising agents proposed so far are phenyl isocyanate [80,81], 1-anthroylnitrile [82], 3,5-dinitro-benzoyl chloride [83], naphthyl isocyanate [84] and naphthoyl chloride [84], However, the lack of fluorescence activity and the need for synthesis through a multistep reaction for some derivatising agents limits their application in a real-world analysis. In fact, only a few of them were applied in the determination of AEOs in environmental samples. Zanette et al. [84] developed derivatisation and separation... 

Isocratic method. Whenever possible, use isocratic HPLC condition, as this is affected less by the variation in flow rate, temperature, and dwell volume. If gradient HPLC conditions have to be used, a simple linear gradient is preferred over multistep gradients. Complicated gradient conditions are more susceptible to differences between HPLC instruments (e.g., flow rate, dwell volume). 

The selectivity of cyclodextrins toward the various molecules is not high enough to attain complete (or acceptable) separations by one-step operations. Enrichment, of one component or partial separation of various components of a mixture can be attained relatively easily. However using cyclodextrins in multistep processes, i.e. the various chromatographic techniques, very effective separations can be achieved. Particularly in RP-HPLC the application of immobilized CDs and CDs dissolved in the mobile phase became one of the most promising methods. 

The probable reaction mechanism of this multistep reaction is given in Scheme 1 above (see the section Preparation of S15 ). The product was obtained as pale-yellow crystals in 8% yield, sometimes still containing traces of Sio which can be removed by recrystallization from CS2. The largest sulfur ring detected by HPLC in this reaction mixture is S30 which has however not been isolated yet [14]. 

In summary, we have demonstrated the potential utility of preparative reversed phase HPLC as a tool to purify fermentation products in multigram scale on laboratory instruments. Secondly, due to its intrinsic selectivity and compatibility with the use of high flow rates, RPHLC is a useful tool for rapidly purifying crude fermentation extracts to a level of purity equivalent to that obtained by multistep time consuming procedures. We... 

The activation of DNT has been shown to be a multistep process involving metabolism in the liver, excretion into the bile, deconjugation of metabolites and further metabolism by the intestinal flora, re-uptake (enterohepatic transport) of metabolites into liver, and finally activation and binding to cellular macromolecules in the liver [56], More recent studies [57] involving rats pretreated with coal tar creosote, which potentiates the genotoxicity of 2,6-DNT, elucidated a complex interaction that balances metabolic activation, uptake, and detoxification. The study monitored intestinal flora enzyme activities, bacterial analysis, mutagenicity of urine samples, HPLC analysis, and hepatic DNA adducts over a five-week exposure period. The location of nitroreductase activity was an... 

Another example of a multistep synthesis using only PSQ purification procedures was described by Hodges etal. In the first step, 1,3-diketone 47 was reacted with an excess of hydrazine 48 in the presence of morpholinomethyl-polystyrene (49). Removal of excess 48 by quenching with polymer-bound isocyanate 50 afforded pyrazole 51 with a purity of 97% (HPLC) but in quite moderate yield (48%). In the second step, 51 was converted into a mixed anhydride (by treatment with isobutyl chloroformate in the presence of 49) and subsequently transformed in situ into 5 3 by reaction with an amine. Addition of the polymer-bound reagents 50 and 52 removed excesses of chloroformate and amine by filtration and 53 was isolated in 75% yield with a purity of 97% (HPLC). 

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