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Anal. Chem. 1980, 61, 228-232
Capillary Isotachophoresis/Mass Spectrometry Harold R. Udseth, Joseph A. Loo, and Richard D. Smith* Chemical Methods and Separations Group, Chemical Sciences Department, Pacific Northwest Laboratory, Richland, Washington 99352
The on-llne comblnatkm of caplliary isotachophoresk (CITP) wlth mass spectrometry Is demonstrated for the first tlme. The CITPIMS interface lo based upon electrospray lonlzation and Is ldentlcal wlth that developed previously for caplllary zone Wrophoresis (CZE)/MS. Separatkns were conducted In untreated 100 pn 1.d. fused sl#ca capllarles havlng lengths of 0.6-2.5 m, at voltages up to 35 kV. The method involves elution of the leading electrolyte to the electrospray source followed by a sequence of separated analyte bands (If sufficient tlme Is provided for development) and, finally, the tratllng electrolyte. The CITP/MS was demonstratedto aUow very hlgh resolullon separations of quaternary phosphonium ions and other ionic substances havlng very small dlfferences in electrophoretic mobilities. Nearly ideal band shapes are obtained in most separatlons derplte the presence of eiectroosmotic flow. The pdentlai for applkation to very dllute sample sdutlons is demonotrated by detection of analytes havlng lo-@M concentratlons, wlth signal to noise ratios of approximately lo2 for 801118 components, whkh Is at least 2 orders of magnitude better than CZEIMS. CITPIMS appears to be an attractlve complement to CZE/MS for dilute (low lonlc strength) eokrtkns slnce much greater sample slzes can be addressed wlthout loss of efficiency.
INTRODUCTION Capillary zone electrophoresis (CZE) has been demonstrated to have tremendous potential for high-resolution separations of very small samples. Previously we have described the direct combination of capillary zone electrophoresis with mass spectrometry (CZE/MS) based upon an electrospray ionization (ESI) interface (I, 2). More recently, interface developments were reported that provide significantly improved flexibility with regards to CZE buffer and ionic strength (3). A potential limitation of CZE/MS for some applications, where both high sensitivity and high efficiency separations are required, stems from the small sample capacity (typically 1-50 nL). In addition to the requirement of a small capillary diameter (loo) than that which can be tolerated in CZE. Second, CITP generally results in concentration of analyte bands (depending upon concentration relative to the leading electrolyte), in contrast to the inherent dilution obtained with CZE. A third benefit of CITP is derived from the continuous elution of sample bands. As pointed out by Giddings (IO, I I ) , the chromatographic peak capacity needed to separate all the components of a sample greatly exceeds the number of sample components. In contrast, with isotachophoresisone sample band immediately follows another and detector time is not wasted (once a separation is obtained). Finally, samples elute during CITP in a manner especially well suited to mass spectrometric detection. Ideal CITP bands are flat topped, and the scan speed of the mass spectrometer need not generally be challenged by the dynamic nature of “sharp” chromatographic peaks. In addition, all bands have similar ion concentrations so that one would not expect excessively large differences in signal intensities between bands. (Thus, a broad dynamic range detector is generally not required.) Similarly, additional information on concentration is conveyed by the length of the analyte band. In this report, we describe the fist use of combined on-line CITP/MS and its complementary role to CZE/MS. We show that CZE/MS instrumentation can be used for CITP/MS without major modification, that the strong electroosmotic flow does not degrade separations, and that both high sensitivities and high-resolution separations can be obtained.
EXPERIMENTAL SECTION Instrumentation. The details of the mass spectrometer, capillary electrophoresis system and electrospray ionization (ESI) interface have been described elsewhere (1-3). A 100 wm i.d. untreated fused silica capillary column was used to effect CITP separations. The inlet of the capillary was biased to the desired potential by an electrode immersed in a buffer solution. The exit end of the capillary has an electrical contact made through a flowing liquid sheath electrode (3). This contact is continuously renewed and mixes with the column eluent to form the solution, which is immediately electrosprayed (3). The solvent is removed from the electrospray plume by a countercurrent flow of warm 0 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 3, FEBRUARY 1, 1989
(-60 O C ) nitrogen. The nozzle-skimmer inlet (ion sampling region) of the mass spectrometer is located approximately 2 cm from the capillary terminus. The mass spectrometer, assembled from Extrel Corp. (Pittsburgh, PA) components, has three differentially pumped chambers. The first is between the nozzle and skimmer, the second contains a rf-only focusing quadrupole, and the third chamber houses a quadrupole mass spectrometer and detector (2, 3). Methods. The CITP/MS sheath flow and cathode voltage (3) were adjusted to form a stable electrospray at the capillary terminus. The column was loaded with the leading electrolyte and the head of the capillary, and the high-voltage electrode (anode for cationic separations)was placed in the sample reservoir. The sample was loaded intothe capillary by electromigration (although both hydrostatic and syringe injection procedures could also be used). When the desired amount of sample had been loaded the voltage was turned off. The high-voltage electrode and capillary inlet were then transferred to the trailing electrolyte reservoir and the voltage was reapplied to begin the separation. The leading electrolyte was 0.001 M ammonium acetate, and the trailing electrolyte was 0.001 M tetraoctylammonium bromide (TOAB). All solutions were prepared in 1:l water/methanol. The ammonium acetate (reagent grade) was obtained from Spectrum Chemicals. The quaternary ammonium and phosphonium salts were analytical grade and obtained from Aldrich. Dopamine, epinephrine, and DL-aminO acids were purchased from Sigma Chemical Co. The methanol was HPLC grade from J. T. Baker. All were used without further purification. The water was deionized and doubly distilled.
(CH2sCH M/Z)(C6H5)3 289 P+
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Contrasts with Conventional Isotachophoresis. The arrangement for CITP/MS was unusual in several respects compared to conventional practice in isotachophoresis (6-8). One objective of this study was to determine to what degree these differences might degrade isotachophoretic separations. The relatively rapid migration of the bands in the capillary due to electroosmosis accounts for one major difference. As the leading electrolyte elutes the solute bands and trailing electrolyte fill the capillary, the electrical resistance in the capillary steadily increases. The isotachophoretic separation and electroosmotic flow occur concurrently such that the lead electrolyte will elute followed by the solute bands and, finally, the trailing electrolyte. Thus, the electroosmotic velocity will initially be characteristic of the leading electrolyte and gradually transforms to that expected for the trailing electrolyte. The disappearance of the leading electrolyte from the capillary terminus was not expected to affect the separation. When the first analyte band begins to elute, if fully separated, the steady state obtained in the capillary will be equivalent to one in which this solute was used as the leading electrolyte (at a concentration defined by the original leading electrolyte). If the first band is not fully separated upon reaching the capillary terminus, subsequent trailing bands could continue to develop. Of practical significance for CITP/MS is the stability of the electrospray as various solute bands elute. The presence of electroosmosis is often regarded as undesirable in isotachophoresis not only because it requires the use of a longer column but also because of potential degradation to the band profiles. With traditional detectors band identification and quantitation are dependent on obtaining good band shape (Le., sharp, flat-topped bands). Since the samples elute in a continuous sequence of band there are no "gaps" between components. If the individual bands are not sharp, the signal from conventional detectors (based upon conductivity or temperature, but not necessarily W detection) would tend to degrade to a featureless ramp. It was anticipated that the selectivity of mass spectrometric detection would relax requirements for band sharpness. The different mass spectrum for each component should readily allow de-
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RIE CITP-MS
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RESULTS AND DISCUSSION
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50,indicating that substantially lower concentrations could be detected. The tetramethylphosphonium ion, which apparently has an electrophoretic mobility insufficiently different from ammonium ions in the leading electrolyte to be isotachophoretidy separated, has a peak shape and width more typical of a CZE separation. The combination of preconcentration during injection and isotachophoretic analysis
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TIME (MINUTES)
Figure 6. CITP/MS separation of a mixture of dopamine and two amino acids in a 2.15-m capillary showing the single ion plots, the trailing electrolyte, and the RIE.
forces the other sample components into a relatively narrow band. The narrow width at half height for mjz 259 and 291 (
