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A new design for large, dilute sample loading in capillary electrophoresis.

In this paper, we report on a new design for a fully in-line sample preconcentration for capillary zone electrophoresis (CZE). It comprises a miniaturized (50-75 microns i.d. x 1 mm) reversed-phase C18 bed (2-4 nL) connected without any dead volume to a CZE capillary. The system permits the fast loading of relatively large sample volumes into the CZE column after elution of the trapped analytes with a small organic solvent volume (20-50 nL). The trapping capacity for peptides was in the high picomole range. No breakthrough or other losses were observed either during microliter-range injections or subsequent washings. The trapped analytes were eluted into the CZE column with less than 50 nL methanol/water (80:2 vol/vol). The narrow band in which samples enter the CZE columns allows direct CZE separation without the necessity of refocusing or stacking. The system performance was studied in relation to the volume and concentration of the leading stacking buffer using electrospray ionization (ESI)-compatible electrolytes. In addition, the system allows fractionation of the trapped analytes using subsequent stronger elutropic elution organic solvent/water mixtures, resulting in a semi-2D separation technique. The system was applied to the analysis of protein digest mixtures and to extremely complex major histocompatibility complex (MHC) class I associated peptides.