Anal. Chem. 1998, 70, 3003-3008
Separation of Tryptic Digests Using a Modified Buffer in Pressurized Capillary Electrochromatography with an Ion Trap Storage/ Reflectron Time-of-Flight Mass Spectrometer Peiqing Huang, Jing-TaoWu,† and David M. Lubman*
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055
A rapid and efficient separation method using pressurized capillary electrochromatography (pCEC) has been developed to separate protein digests. The effects of mobilephase 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 β-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. Capillary electrochromatography (CEC) is a developing separation method, wherein a high voltage is applied to a capillary LC column which induces electroosmotic flow (EOF) to drive the mobile phase through the column. CEC couples the very high efficiency of capillary zone electrophoresis with the high selectivity of reversed-phase LC and has the potential to become a powerful separation tool for the analysis of complex mixtures. CEC has been performed by several groups for analysis in a variety of applications.1-4 It has been used to separate polycyclic aromatic hydrocarbons, pharmaceutical compounds, amino acids, peptides, and oligosaccharides. The primary difficulty in CEC has been associated with bubble formation produced by Joule heating as a result of the electric current passing through the column. The bubbles generate baseline noise and make it difficult to obtain stable flow conditions. There are two approaches to minimize bubble formation. In one method, the buffer vials at both the inlet and outlet ends of the capillary are pressurized by using an inert gas such as nitrogen.5 Alternatively, an HPLC pump is coupled to the inlet end of the † Current address: Du Pont-Merck Pharmaceutical Co., P.O. Box 30, Building 115, Newark, DE 19714. (1) Yan, C.; Dadoo, R.; Zare, R. N. Anal. Chem. 1996, 68, 2726-2730. (2) Colon, L. A.; Guo, Y.; Fermier, A. Anal. Chem. 1997, 69, 461A-467A. (3) Huber, C. G.; Choudhary, G.; Horvath, C. Anal. Chem. 1997, 69, 44294436. (4) Schmeer, K.; Behnke, B.; Bayer, E. Anal. Chem. 1995, 67, 3656-3658. (5) Smith, N. W.; Evans, M. B. Chromatographia 1994, 38, 649-657. (6) Behnke, B.; Bayer, E. J. Chromatogr. 1994, 680, 93-98.
S0003-2700(98)00124-3 CCC: $15.00 Published on Web 05/21/1998
© 1998 American Chemical Society
capillary to provide supplementary pressure.6-8 This approach is more desirable in most applications since it can be conveniently used to fill the capillary column with solvent and to flush the column. Also, the gradient elution necessary for separating complex mixtures can be easily realized. This pressurized capillary electrochromatography (pCEC) is a hybrid form of capillary LC and CEC. In this method, a pressure is applied to a capillary column along with an electric field. The overall velocity of the mobile phase is contributed to by the pressure and EOF, so that a relatively high analysis speed can be achieved. The electric field also causes different electrophoretic migration for charged analytes, so that selectivity can be tuned by varying the electric field strength for charged analytes. Pure CEC has been used to separate neutral analytes such as polycyclic aromatic hydrocarbons, while pCEC has been used mostly to separate charged analytes such as alkaloids, oligonucleotides, and peptides. In pCEC, the pressure is not only used to minimize bubble formation, but it also provides a means of tuning selectivity for the separation of charged analytes by varying the electric field,6,9-11 which cannot otherwise be achieved in pure CEC. The separation of peptide mixtures by pCEC-MS has been reported in recent work by our group.11,12 Highly efficient separations of protein digests in
