Pressurized capillary electrochromatography (pCEC) has been coupled to an ion trap storage/reflectron time-of-flight mass spectrometer for the analysis of peptide mixtures and protein digests. Taking advantage of the electroosmotic flow, high separation efficiency has been achieved in pCEC due to a relatively flat flow profile and the use of smaller packing materials. A supplementary pressure was used in these experiments which suppressed bubble formation and also allowed the tuning of the elution of peptides using the electrical field. In this work, a fast separation of a six-peptide mixture has been successfully performed. Using columns only 6 cm long, a tryptic digest of bovine cytochrome c was fully separated in around 14 min by properly tuning the applied voltage and the supplementary pressure. In addition, relatively complex protein digests, such as a tryptic digest of chicken ovalbumin, were analyzed using this pCEC/MS system, and more than 20 peaks were resolved in the total ion current chromatogram within 17 min. The use of an ion trap storage/reflectron time-of-flight mass spectrometer as an on-line detector further increased the resolving power of the pCEC by unambiguously identifying coeluting components. The nonscanning property of the time-of flight mass analyzer and the ion signal integration capability of the ion trap were successfully combined to provide rapid and sensitive full-mass range detection in these experiments.

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A mixed-mode (reversed-phase/anion-exchange) stationary phase has been used as the capillary column packing for investigation of the separation of peptide mixtures in pressurized capillary electrochromatography (pCEC). This stationary phase contains both octadecylsilanes and dialkylamines. The amine groups of the stationary phase determine the charge density on the surface of the packing and can produce a strong and constant electroosmotic flow (EOF) at low pH. A comparison was made in terms of the capability of separating tryptic digests between the mixed-mode phase and C18 reversed phase. In addition, the constant EOF enabled the tuning of the retention and the selectivity of the separation by adjusting the mobile phase pH from 2 to 5. Furthermore, the magnitude and the polarity of the electric voltage were demonstrated to greatly influence the elution profiles of the peptides in pCEC. An ion trap storage/reflectron time-of-flight mass spectrometer was used as an on-line detector in these experiments due to its ability to provide rapid and accurate mass detection of the sample components eluting from the separation column.

A rapid and efficient separation method using pressurized capillary electrochromatography (pCEC) has been developed to separate protein digests. The effects of mobile-phase ion conductivity, pH, and column size on separation speed and column efficiency were studied and optimized. The pCEC method was demonstrated to provide enhanced speed with more efficient and selective separation than HPLC by performing separations of a bovine beta-lactoglobulin A digest. A human hemoglobin digest was separated by pCEC under optimized conditions, and more than 20 peaks were separated in less than 20 min. Using an ion trap storage/reflectron time-of-flight mass spectrometer, coeluting components were clearly identified, and the resolving power of the pCEC method was further enhanced.