Head-Column Field-Amplified Sample Stacking in Binary System Capillary Electrophoresis:  A Robust Approach Providing over 1000-Fold Sensitivity Enhancement

Abstract

Effective electrokinetic field-amplified sample injection occurring at the capillary inlet from a sample volume equivalent exceeding that of the capillary up to 10-fold is described and demonstrated to provide over 1000-fold sensitivity enhancement. Successful application of this head-column field-amplified sample stacking approach to the analysis of positively chargeable, hydrophobic compounds in binary system capillary electrophoresis is shown to require an initially introduced low-conductivity zone (water plug) of >1 mm length, a sample injection voltage <20 kV, and an injection time interval <60 s. Following these conditions for more than 1500 runs with capillaries of 50 μm i.d. and about 20 cm effective length, damaging heat production during electroinjection within the low-conductivity zone at the column inlet (boiling of solvent and possible deposition of solutes or fusing of capillary walls) could be prevented. The solute amount injected by head-column field-amplified sample stacking is further shown to be dependent on the organic fraction and the buffer in the sample solution. High content of organic solvent, low conductivity, and the presence of a small amount of H(+) (50-100 μM) provides the highest sensitivity for analysis of positively chargeable model substances, including amiodarone and desethylamiodarone. Solutes present at the nanomolar level can thereby be accumulated from a sample volume equivalent of about 4 μL (with injection of about 20 nL of sample solvent into the capillary) and measured by UV absorption detection. To prevent disturbances caused by electrolysis, sample vials should be employed only once. The data obtained further show that quantitation can be reliably performed using internal calibration based on peak height (RSDs for inter- and intraday determinations are on the 2% level). However, due to variation of the roughness of the capillary walls and cuts, the time interval between operational steps, and trace adsorption onto the capillary walls, the length of the water zone drawn by capillary action on the inlet side is not constant, and external calibration therefore cannot be employed for quantitation.

Analysis of the disaccharides derived from hyaluronic acid and chondroitin sulfate by capillary electrophoresis with sample stacking

“…Using hydrodynamic sample injection, the detection sensitivity was found to be more than ten-fold worse (data not shown). Furthermore, field-amplified sample injection with application of a water plug prior to electroinjection from the sample dissolved in a low conductivity environment (water), as was previously documented to provide over 1000-fold sensitivity enhancement in other configurations [41], provided higher peaks for di-CS and di-HA (data not shown). This approach, however, was not effective with samples of high ionic strength, including the serum samples prepared in this work, and was thus not further investigated.…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…As analytes reach the sample matrix/BGE interface, their velocities decrease, resulting in a stacking of the analytes into narrow bands [183]. In CZE applications utilizing FASS, studies have reported on a doubling of sample stacking efficiencies [185] and 1000-fold increases in sensitivity [186], resulting in considerable improvements in loadability over conventional CZE.…”

High‐sensitive protein analysis by FESI‐CE‐MALDI‐MS

“…Several electrophoretic-based preconcentration methods such as large-volume sample stacking (LVSS) [9], fieldamplified sample stacking (FASS) [10] and field-enhanced sample injection (FESI) [11,12] have been developed. These techniques are based on the reduction of the analyte migration velocities when they encounter a drop in electrical field strength at the boundary between the sample matrix and BGE zones.…”