Anal. Chem. 1995,67,3656-3658
Capillary Electrochromatography-Electrospray Mass Spectrometry: A Microanalysis Technique Karl Schmeer, Beate Behnke, and Emst Bayer* lnstitut fur Organische Chemie, Universitat Tubingen,Auf der Morgenstelle 18, 72076 Tubingen, Germany
An electrospray mass spectrometer was coupled to an
capillary electrochromatographcsystem. Peptides were analyzed in a capillary column packed with 1.5-pm reversed phase stationary phase at flow rates of 1-2 p V min. Supplementary pressure was applied to stabilize the electrochromatographicperformance. The interface employed allowed fast and simple installation of the electrochromatographic system to the mass spectrometer, requiring neither sheath flow nor long transfer capillaries. This CEC-MS coupling allows the routine detection of picomole quantities. Capillary electrochromatography'-" can be considered as a variant of reversed phase liquid chromatography performed in packed capillary columns. An electric field is applied across the length of the columns to transport the eluent by electroosmosis. The velocity of the electroosmotic flow is independent of the particle size of the stationary phase over a wide range, thus allowing the use of columns packed with 1.5pm particles up to a length of 0.5 m. With such columns, efficiencies of more than 200 000 plates/column can be comparable to those obtainable in capillary zone electrophoresis (CZE) and thus exceeding by far the efficiency of 30 OOO plates generally obtained in HPLC. The use of such small particles in pressure-driven HPLC requires uncomfortably high pressure. Separation of hydrophobic analytes is easily performed in capillary electrochromatography (CEC) with up to 90%modifer, typically acetonitrile. An instrumentation for gradient elution in CEC has been described recently' which allows very flexible tuning of the selectivity analogous to its effect in HPLC. In addition, the selectivity in analysis of charged analytes can be increased by electromigration of the sample molecules comparable to CZE. To minimize Joule heat, CEC columns with inner diameters of 50-100 pm are generally applied. The flow rate of up to 2 pL/min and loadability of sample quantities in the nanogram range are considerably higher than in CZE, where flow rates in the nanoliter per minute and sample amounts in the picogram range are common. In HPLC, the inner diameter of the columns can be varied over a wide range and correspondingly also the flow rate and loadability. Capillary electrochromatography offers the possibility of sample preconcentration from diluted solutions (1) Knox, J. H.; Grant, J. H. Chromatographia 1991,32,317-328. (9Yamamoto, H.; Baumann, J.; Emi, F. J. Chromatogr. 1992,593,313-319. (3) Smith, N. W.; Evans, M. B. Chromatogruphia 1994,38,649-1357, (4) Behnke, B.; Bayer, E. J. Chromutogr. A 1994,680,93-98. (5) Tsuda, T. LC-GC 1992,5,26-36. (6) Behnke, B.; Bayer, E. J. Chromatogr., in press. (7) Verheij, E. R.; Tjaden, U. R.; Niessen, W. A. M.; van der Greef, J. J. Chromatogr. 1991,554,339-349.
3656 Analytical Chemistry, Vol. 67, No. 20, October 15, 1995
analogous to HPLC. This is especially convenient in the gradient elution mode. A major drawback of pure CEC was the difficulty of obtaining stable flow conditions. As recently demonstrated, this problem is readily overcome by using supplementary pre~sure.~-6 Electrochromatography provides several advantages for coupling to the mass spectrometer. With capillary columns of 100pm i.d., flow rates of 1-2 pL/min are obtained, a value almost ideal for electrospray-MS. Therefore no interface like a liquid sheath flow analogue to CZE is required. Sintered silica gel frits allow the direct coupling of the packed capillary columns without additional transfer capillaries. The spray is thus formed directly at the outlet side of the column. On-line coupling of micellar electrokinetic capillary chromatography (MECC) is usually restricted to MS detection due to incompatible amounts of compounds necessary for micelle formation, typically anionic surfactants. A coupling method for hydrophobic analytes in electrodriven techniques has been missing. CEC is closing this gap. A technique related to CEC is pseudoelectro~hromatography.~~~ Here the eluent is transported by pressure analogous to conventional HPLC, and therefore, the same restrictions to column length, particle size of the stationary phase, and efficiency apply. An electrical field is used for tuning the selectivity in separations of charged analytes. The first coupling of a pseudoelectrochromatographic system to a FAB-MS was carried out by van der Greef et al. in 199L7The columns used had an inner diameter of 220 pm. These large inner diameters were necessary to provide a flow rate of 5-15 pWmin suitable for the CF-FAB interface. Coupling of a pseudoelectrochromatographic system with columns of the same internal diameter to an electrospray-MS with a sheath flow interface was also demonstrated by the same group in 1993.* A coupling of pure electrochromatography to FAB-MS was reported in 1994 by Gordon and Lord.g They presented a separation of steroids in a capillary column of 50-pm i.d. packed to a length of 35 cm with 3;um particles. In all approaches published so far,7-9transfer capillaries were required to connect the separation columns with the mass spectrometer resulting in loss of efficiency. In this paper we present and discuss the fist coupling of electrochromatography with supplementary pressure to an electrospray-MS. As will be demonstrated, the eluent is mainly transported by the electroosmotic flow and pressure is only employed to stabilize the EOF at high electrical field strength. (8) Hugener, M.; Tinke, A. P.; Niessen, W. M. A,;Tjaden, U. R.; van der Greef. J. J. Chromatogr. 1993,647, 375-385. (9) Gordon, D.B.; Lord, G. A. Rapid Commun. Mass Spectrum. 1994,8,544548.
0003-2700/95/0367-3656$9.00/0 0 1995 American Chemical Society
The combination of packed capillary columns and interface described provides an optimum of efficiency and sensitivity. MATERIAL AND MEIUOW Preparation of the Packed Capilby cohunns. Fused-silica capillaries of 1Wpm i.d. and 3Wpm 0.d. were obtained from Polymicro Technology (Phoenix AZ). The frits consisted of sintered fused-silica gel (Gromsil, 4 = 5pm; Grom, Hemenberg, FRG) as described in ref 4. The capillary columns for coupling to the mass spefbometer were made by first sintedng a frit on the outlet side, slurry packing with reversed phase silica gel (Gromsil ODS2, 4 = 1.5 pm; Grom), and then preparing of the inlet frit. The capillary columns for the W detection mode were produced as described in ref 4. Apparatus. The eleetrochromatographic system consists of a modular capillary electrophoresissystem (Gmm) combined with a standard HPLC system (Sykam, Gilching. FRG). A Chromapac GRGA (Shimadzu,Kyoto, Japan) was used for data pmcessing. Solvent splitting is accomplished by a stainless steel T-piece and a resistance capillary of Wpm i d . and 5Ocm length. A split ratio of -12oOo is obtained at zero electric field strength. The eluent is filtered by a guard column before entering the injection dwice. The T-piece is grounded to protect the HPLC system from possible damage caused by the high voltage. Injection of -0.1 pL was accomplished by filling the eleetrolyte reservoir on the inlet side with 5 pL of sample and pressurizing for 10 s at 200 bar. The reservoir was then 0ushed with eluent. El-matcgraphy. For the on-line peptide separation, a chromatographic column of 2 k m overall length and l W p m i.d. was packed to a length of 19 cm with 1.5pm reversed phase particles. The length of the frits was 0.3 cm each. The supplementary pressure was 180 bar at 0.5 mWmin flow rate of the pump. Separation of peptides in conventional HPLC is us& carried out with aceonitrile water mixtures containii 1 mWL hifluoroacetic acid ITFA). To avoid bubble formation resulting fromexcessive Joule heating, the TFA concentration was reduced to 0.07 mWL The eluent was acetonitrile-water TFA (So: 20). The concentrations of the peptides were 20 pg/mL each in acetonitrile-water (4060). Dissolution of anaiytes in a solvent of lower elution shngth than the eluent results in focusing of the sample at the beginning of the stationary phase. Eleetrochm matography was performed at 20.2 kV and 0.8 pA. The applied potential gradient is given by the difference of +25 kV of the power supply and f4.8 kV of the steel needle. MEW Spedmmeizy. AU mass spectra were recorded on a Sciex API III triple-quadrupole mass spectrometer, having an electrospray ion source (Sciex, Toronto, Canada)and a m/z mnge of 2400 dalton. Calibration was carried out with a Nal solution. Spectra were recorded in positive mode (dwell !he 0.5-1 ms, step size 0.5-1 amu; data acquired to disk). For data acquisition, data processing and the control of the mass spectrometer a Macintosh IIx was employed. The orifice voltage was held at 70 V and the needle voltage at 4.8 kV. EC-MS Interface. The electrochromatographicsystem was connected to the MS so that the outlet of the separation column was positioned within the inner steel needle of the ion source, thus avoiding long connection capillaries Fire 1). The anode was connected to the eleckolyte reservoir on the inlet side. To insulate the mass spectrometer against the high voltage, a Ian-thick tubber mat was attached to the front side of the mass spectrometer and the column was passed through a
+
Power SUPP~Y
Pigum i. Schematic representation of the setup for capillary electrochromatcgraphy-mass spectrometry.
270 bar
I 8
A*
8
04
0
0.5
I
1.5
2
2.5
linear flow velocity (mmls) FlguW 2 Influence of supplementary pressure and electric field strength on the flow velocily: column. fused silica, 1Wpm i.d.. 40cm length, packed for 23 cm. 4 = 1.5pn; buffer, 2.5 mM phosphate, 80% acetonitrile. pH 4 pressure, 80-270 bar with 0.25-1 mumin.
h n n hole therein into the interface. Between the power supply for the needle voltage (of the MS) and the steel needle, a W M Q load resistor was inserted. chemicals. The peptides were synthesized on an AB1 431A peptide synthesizer using Fmoc chemishy. The Fmoc amino acids were purchased from Nova Biochem (Eiufelfmgen,Switzerland) and Rapp Polymere (nrbingen, Germany). All other chemicals were products of Ruka (Buchs, Switzerland) and Merck (Tlarmstadt Germany). AU chemicals for buffer preparation were of research grade. RESULTS AND DISCUSSION In eleckochromatography with supplementary pmsure, the 00w velocity is a function of the electric field shngth and the applied pressure as shown in Figure 2. Surprisingly, the conhibutions of presswedriven and electdriven flow are not additive. With increasing eleCtro0Smotic flow, the iduence of the pressure decreases. At an electric field strength of 100 kV/m, the overall Am/ytkx/Chmlishy, Vol. 67, No. 20. October 15, 1995 36-
7.39min
loo-
RI: 5.58 min
1
75.
3
w,i
[M+Na] 736
' 19.729
1
counts
Y( DCB)-G-G-F-L-OMe
Y(DC6)-G-G-F-L-NH 2
25-
8899 counts
0.0
2s
5,O
7,5
10,o
123
15,O
Time (min)
Figure 3. Extracted mass electrochromatogram of mlz714 and 729 for the on-line peptide separation. Conditions as described in the experimental section.
75
5
I
E 50.
mlz (emu)
flow velocity is thus almost independent of the applied pressure. For this reason, the ion current detected by the MS collapsed immediately when the anode voltage was switched off. The high selectivity and separation power of the electrochromatography is demonstrated with a mixture of enkephalin methyl ester and enkephalin amide (Figure 3). The selectivity of the separation is enhanced by electromigration of the sample molecules. At low pH, the amide carries an additional positive partial charge, migrates faster to the cathode, and is therefore eluted first. The electric field strength of 100kV/m is signiscantly higher than the 20-60 kV/m typically used in capillary electrophoresis. Thus, the separation was strongly influenced by an electrophoretic separation mechanism. Here, the first coupling of an electrospray-MS with an electrochromatographic system in which the flow is mainly carried by the electroosmotic flow is presented. Additional sheath flow, which is usually required in CE-MS coupling, is unnecessary on account of the relatively high flow rate of 1-2 pL/min. In addition, the frit at the end of the column enhances the process of spraying, thus also improving the signal intensities in the MS. Therefore, the focused peak reaches the mass spectrometer undiluted. The absence of transfer capillaries eliminates additional postcolumn dispersion. For these reasons, the mass spectrometer detects very narrow peaks of only 4-8s peak width. The mass spectra of the corresponding peaks (Figure 4) show a high abundance of Na+ and K+ adducts, reducing significantly (10) Wahl, J. H.; Goodlett, D. R ; Udseth, H. R.; Smith, R. D. Anal. Chem. 1992, 64, 3194-3196. (11) Henion, J. D.; Mordehai, A. V.; Cai, J. Anal. Chem. 1994,66, 2103-2109.
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Analytical Chemistty, Vol. 67, No. 20, October 15, 1995
Figure 4. Mass spectra taken from the chromatographicpeaks in Figure 3.
the intensity of the protonated species. In order to detect the small amounts of peptide injected (3 pmol) it was found necessary to decrease the resolution of the MS, thus increasing its sensitivity. The alkali metal ions possibly originate from the underivatized silica spheres in the frits. Deactivation by silanization should prevent this phenomenon. Further improvement of the sensitivity is expected by gold coating and narrowing of the outlet side of the capillary C O ~ U ~ U I . ~ With such measures attomole-level detection limits have recently been obtained in CZE-MS coupling.lOJ1 In summary, the coupling of electrochromatography with electrospray mass spectrometry can be regarded as a very promising method. The small amount of sample and the enormous separation power of the CEC combined with the information provided by the MS represent an excellent combination, especially for microanalysis. ACKNOWLEDGMENT The authors gratefully acknowledge the valuable technical assistance of E. Grom and E. Braun, Tubingen, and thank G. Panhaus and E. Henkel for the synthesis of the peptide samples. Received for review March 23, 1995. Accepted August 2, 1995.e
AC9502864 Abstract published in Advance ACS Abstracts, September 1, 1995.
