SOME PRACTICAL ASPECTS OF UTILIZING THE ONLINE COMBINATION OF ISOTACHOPHORESIS AND CAPILLARY ZONE ELECTROPHORESIS

The on-line combination of isotachophoresis (ITP) and zone electrophor esis is a very effective tool for increasing the separation capability and sensitivity of capillary zone electrophoresis (CZE). Its most eff ective version is performed in column coupling instrumentation and is characterized by isotachophoresis in the first capillary serving as th e efficient preseparation and concentration stage followed by on-line transfer of the sample cut into the second capillary where analytical zone electrophoresis proceeds as the second stage. The on-line transfe r of the sample from the first capillary into the second is always acc ompanied by the segments of some additional amounts of the leader and/ or terminator from the ITP step. This results in transient survival of isotachophoretic migration during the second stage. Hence the separat ion process during the second stage can be characterized as the destac king of analytes followed by zone electrophoretic separation. In this paper, both a theoretical and an experimental study is presented showi ng that destacking of analytes is a selective process, which affects s trongly the most important final analytical parameters of the detected zones, namely the detection time and zone variance, and thus it makes the simple use of detection data for qualitative analysis misleading. It was shown that both the detection time and variance of a zone of a n analyte depend strongly on the amount of the accompanying segments o f the leader/terminator transferred and on the actual type of electrol yte system selected from the generally possible types. These types are (i) L-S-L, where the leading electrolyte (L) from ITP stage serves as the background electrolyte (BGE) during CZE (S = sample), (ii) T-S-T, where the terminating electrolyte (T) from the ITP stage serves as th e BGE during CZE, and (iii) BGE-S-BGE, where L, T and BGE are mutually different. Explicit equations were derived enabling one to predict mi gration time and zone variance for actual working conditions, and, for model systems, the theoretical data were calculated and verified expe rimentally. Further, it is shown that the systems T-S-T and L-S-L are user friendly and a simple standardization procedure was proposed, all owing correct qualitative evaluation of the analytical data in these s ystems. Finally, a theory is presented predicting the existence of ano malous variances of zones in the record of analysis in BGE-S-BGE syste ms and its experimental verification is given.