Multistep elution

Tong, W., Link, A., Eng, J. K., and Yates, J. R., Identification of proteins in complexes by sohd-phase microextraction/multistep elution/capiUary electrophoresis/tandem mass spectrometry. Anal. Chem., 71, 2270-2278, 1999. 

Although multistep affinity assays with redox-labeled targets have been described (Wang et al. [117]), most of the assays use enzyme-labeled species in conventional indirect formats (competitive, non-competitive). Direct EILAs based on multistep electrochemical affinity assays have also been developed with excellent results. In all these cases the MIP is used to extract the analyte from the sample and, after elution, the analyte is carried on to the electrochemical flow-through cell for being detected. 

Reiger and Ballschmiter [43] described a multistep method for PCA analysis in sewage sludge by cyclohexane-isopropanol extraction and cleanup on silica gel column chromatography. Fractionation on silica gel was achieved by eluting with hexane (FI), which desorbed hexachlorobenzene, 4,4 -DDE, PCB, PCDD, and PCDF. PCAs were then desorbed from the column with (90 10) hexane/di-ethyl ether. The recovery of PCAs by this method was 86%. These authors also noted that cleanup chromatography on activated alumina should be avoided because PCAs were either totally or partially destroyed by dehydrochlorination during the adsorption process. 

The results of Fig. 14 suggested that the purification of the desired product would be best achieved by a multistep approach, i.e., an initial HILIC/CEC step followed by RPC of the desired peptide product. Another example of the resolving capability of the multistep HlLlC/CEC and RPC protocol is illustrated in Fig, 15, which outlines the purification of a synthetic 21-residue amphipathic a-helical peptide from the peptide crude mixture. From the RPC elution profile (top) of the crude peptide, a major peak is obtained with a number of more hydrophilic and hydrophobic impurities, some of which are eluted very close to the main component. Subsequent attempts to improve the isolation of this main peak by varying the pH and/or the ion-pairing reagent used in the RPC mobile phase were unsuccessful, thus ruling out the possibility of an efficient one-step purification by RPC, Application of mixed-mode HILIC/CEC to the crude mixture resulted in the middle elution profile, which shows a major peak (P) as well as several other smaller, yet substantial, peptide components. Collection of the major peak followed by RPC produced the bottom elution profile, with the... 

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