Richard D. Smith, Jon H. Wahl, David R. Goodlett', and Steven A. Hofstadler Chemical Methods and Separations Group Chemical Sciences Department PacificNorthwest Labratoy Richland, WA 99352 Many of the most difficult chemical, environmental, biochemical, and biomedical analytical problems reqnire a combination of instrumental attributes, including speed, low detection limits, wide linear dynamic range, good sensitivity, and high selectivity. For such demanding applications, the on-line combination of separation methods with MS often provides the most practical or perhaps the only approach. The orthogonal nature of the selectivities provided by a chromatographic or electrophoretic separation in conjunction with MS has long been considered attractive. Indeed, GC/MS is firmly established as a definitive analytical technique for many environmental and clinical analyses. The hallmarks of GClMS are its speed, selectivity, and sensitivity. Unfortunately, however, both GC and t h e conventional ionization methods used in MS (primarily electron impact and chemical ionization) reqnire sample volatilization. Thus, GClMS is not amenable to many analytica! problems without invoking often eomplex and problematic chemical degradation o r derivatization procedures designed to modify sample components to "GC-able" forms. Interest in LCIMS has continued to grow, and the technique has begun to open new avenues for the characterization of biological and biomedical samples (I). The 1980s saw the genesis and rapid development of a high-resolution separation method, capillary electrophoresis (CE), primarily because of the efforts of J. W. Jorgeneon of the University of North Carolina (2).He and his co-workers have
' Current address: Immunobiology Research Institute. Route 22 East, P.O. Bar 999,Annandale, NJ 08801 574 A
demonstrated that CE can generate both rapid and very high resolution separations, based on differences in t h e electrophoretic mobilities of charge-carrying species in a n electric field, in small-diameter fusedsilica capillaries. The advantages of the capillary format for electrophoresis are multifold. First, small-diameter capillaries (generally 50-100-pm id.) generate less Joule heat and dissipate this h e a t more effectively, allowing higher electric fields t h a n can be used with conventional electrophoresis and providing faster and higher resolution separations. Second, the capillary format allows for easy au-
REPORT tomation of sample handling and injection. The CE format allows ready implementation of a range of oncapillary detection methods; most effectively and broadly used are UV absorption and fluorescence emission detectors. Since the first commercial CE instruments appeared in the late 19808, CE technology and its applications have grown explosively. In fact, the rate of growth, use, and commercial implementation has wnsiderably exceeded that seen earlier for LC methods. Improvements in injection methods, detector Sensitivity, capillary surface deactivation, and coating technologies, as well as the introduc-
ANALMICAL CHEMISTRY, VOL. 65, NO. 13, JULY 1.1993
tion of new electmphoretic buffer systems, continue to drive further developments in CE for chemical, biological, and environmental applications. The growth of CE as a viable analytical tool is primarily the result of advances in detection methods and a n increasing recognition of its unique capabilities. CE would not be practical without the sensitivity improvements that have been demonstrated with on-capillary UV and fluorescence detection. Detectable amounts in t h e femtomole (lo-" mol) range can be obtained routinely, although optimized and specialized detection schemes have been reported for which detectable amounts extend to attomole (lo-'' mol) and zeptomole (lo-" mol) levels. Thus, for CE with typical capillary diameters, in which effective injection volumes are generally in the range of 1-10 nL, routinely detectable concentrations are typically on the order of lo-" M for the injected sample. Specialized detection systems allow these detectable concentrations to be extended to < lo-'' M, which is well into the regime of trace analysis. Improved detection limits can be obtained by using electrophoretic methods to concentrate sample wmponents during injection. The ability to manipulate and inject extremely small sample volumes, steps that are generally problematic with LC, provides a basis for using CE to confront extreme analytical challenges (e.g., the analysis of, or sampling from, single biological cells). In addition, CE has the flexibility provided by a range of formats (free-zone electrophoresis, electrokinetic micellar chromatography, isotachophoresis, gel electrophoresis, etc.) and a plethora of methods for manipulating injection conditions and separation specificity. Moreover, methods have been developed or are being investigated for CE application to the analysis of practically any substance that can be dissolved or suspended in a liquid. Finally, from a pragmatic viewpoint, the small sample, buffer, and waste volumes required and generated by CE are much less than those used by 0003-2700193/0365-574Ai$04.00/0
1993 American Chemical Society
LC methods; small volumes are attractive because of the trend toward treating all LC (and CE) effluents as hazardous wastes that require expensive tracking and disposal.
Development of CWMS The early work of Jorgenson and coworkers clearly demonstrated the potential power of CE methods, but combining them with MS required the solution of several conceptual and practical problems. First, all early CE experiments, and nearly all today, involve placement of both ends of the capillary into reservoirs of the conductive buffer, where electrical contact is established to defme the CE field gradient. Second, any CE/MS interface must be compatible with low CE flow rates (5 1 FL/min a t most) and should not induce a pressure-driven (laminar) flow in the capillary t h a t would degrade separation quality. Moreover, detection sensitivities must extend to subpicomole levels for CElMS to be of practical value. The early 1980s saw the introduction of the thermospray interface for LClMS ( I ) , but this approach was impractical for CE because of the necessary flow rates (z 100 pL/min) and inadequate sensitivity. In 1984 Fenn and co-workers presented their initial results on electrospray ionization (ESI) combined with MS (3).In the ESIMS method, liquid solutions are nebulized in a high electric field from the end of a capillary a t flow rates in the 1-10-pLlmin range (4-6). More important, the early ESIMS studies suggested that solute sensitivity was exceptional (4). This work stimulated efforts a t Pacific Northwest Laboratory (PNL) to develop on-line CElMS based on ESI with t h e use of a n electrophoretic capillary, one end of which functioned as the electrospray source rather than being immersed in a buffer reservoir. After constructing ESIMS instrumentation at PNL, results were obtained in 1986 and published in 1987 (7).
a capillary to produce highly charged
ure 2a). With this interfacing method it was necessary to select CE conditions giving rise t o a net electroosmotic flow in the direction of the mass spectrometer, that is, a flow arising at the electric double layer of the capillary surface and creating a flat "plug-like" flow profile away from the point of injection. The metallized capillary terminus also served as the electrospray source by having a 3-6-kV difference in voltage between the terminus and the mass spectrometer sampling aperture, which was 1-2 cm away. In ESI the liquid is nebulized from
droplets, typically only a few micrometers in diameter. The charged droplets drift in the electric field between t h e capillary and t h e mass spectrometer sampling aperture. In transit to the sampling orifice and in transport through the MS interface, the droplets experience conditions that cause evaporation. Because the droplets initially are highly charged and close to the physical limit for their size (the Rayleigh limit), they shrink by evaporation and quickly reach a point at which they shed a portion of their charge. By some still
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