The use of solid phase concentrators for on‐line preconcentration of metallothionein prior to isoform separation by capillary zone electrophoresis

Abstract

UV absorbance is a convenient detection method for monitoring a wide variety of different components separated by capillary electrophoresis (CE). However, a significant disadvantage of UV detection is its low sensitivity and this is a major factor limiting the application of CE as an analytical technique. In order to improve sensitivity, several methods of on‐line sample preconcentration by electrofocusing, including “stacking” and isotachophoresis, have been investigated and the potential of on‐line solid phase preconcentrators for this purpose has also been demonstrated. We have developed methods for the separation of isoforms of metallothionein (MT), a low M_r metal‐binding protein which functions as a detoxification system and cellular buffer for heavy metals, primarily in liver and kidney. While a variety of modifications to the capillary surface and electrolyte chemistry have been used to resolve many MT isoforms in liver samples from a variety of species, there are few available alternatives to UV detection and thus sensitivity limits the application of these methods to samples with low MT levels, such as urine or plasma. In this study we have investigated the use of an inexpensive and easy to assemble C₁₈ concentrator which was fitted to the capillary inlet to extend the detection limits for MT isoform analysis. Samples were pressure loaded onto the concentrator for up to 5 min, eluted using 33% acetonitrile and then subjected to electrophoresis in borate or phosphate buffer. The purified isoforms MT‐1 and MT‐2 from rabbit liver were best resolved using an acidic acetonitrile eluent and a phosphate buffer electrolyte at pH 7. Sensitivity enhancement was >700‐fold and the relationship between loading time and isoform absorbance peak area was linear when only a small proportion of concentrator capacity was used. Analysis of sheep liver extracts demonstrated that both sensitivity and specificity were enhanced. The potential of other solid‐phase concentrators is discussed.