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Anal. Chem. 1982,5 4 , 336-338
trode and/or technique with longer time scale is to be employed, the effects of the nonlinear diffusion may increase. For these cases, a more suitable geometry for the upper half of the cell might be a cylinder of diameter comparable to the working electrode to ensure linear diffusion. The present results clearly demonstrate the potential of the cell in situations in which electroactive species have to be measured at concentrations down to the micromolar level and the available sample is very small. Complete coverage of the working electrode limits the working volume to about 10 1L in the present cell. Thus, smaller working electrodes can be employed for analyses in smaller sample volumes. At the millimolar concentration level linear scan voltammetry provides adequate sensitivity and has certain advantages (i.e., speed, precision) over DPV. However, at the micromolar level only DPV offers the required sensitivity.
LITERATURE CITED ( I ) Hubbard, A. T.; Anson, F. C. I n “Electroanalytical Chemistry”; Bard, A. J., Ed.; Marcel Dekker: New York, 1970 Chapter 2.
(2) DeAngelis, T. P.; Bond, R. E.; Brooks, E. E.; Heineman, W. R. AMI. Chem. 1977, 49, 1792-1797. (3) Miller, 6.; Bruckensteln, S. Anal. Chem. 1974, 46, 2033-2035. (4) Eggll, R. Anal. Chim. Acta 1977, 91, 129-138. (5) Messner, J. L.; Engstrom, R. C. Anal. Chem. 1981, 53, 128-130. (6) Huderovi, L.; Stulik, K. Talanta 1972, 19, 1285-1293. (7) Karolcrak, M.; Dreillng, R.; Adams, R. N.; Felice, L. J.; Kissinger, P. T. Anal. Lett. 1978, 9 , 783-793. (8) Ruckl, R. J. Talanta 1980, 27, 147-156. (9) Flke, R. R.; Curran, D. J. Anal. Chem. 1977, 49, 1205-1210. (IO) CaJa, J.; Czerwihki, A.; Mark, H. B.. Jr. Anal. Chem. 1979, 51, 1328-1329. (11) Wang, J. Anal. Chem. Acta 1981, 129, 253-258. (12) Wang, J. Anal. Chem. 1981, 53, 2280. (13) Jarbawl, T. N.; Heineman, W. R.; Patriarche, G. J. Anal. Chlm. Acta 1981, 126, 57-64. (14) Burrows, K. C.; Hughes, M. C. Anal. Chlm. Acta 1979, 710, 255-260. (15) Wang, J.; Ouziel, E.; Yarnitzky, Ch.; Arlel, M. Anal. Chim. Acta 1978, 102,99-112. (16) Adams, R. N. “Eiectrochemlstry at Solld Electrodes”; Marcel Dekker: New York, 1969; Chapter 3.
RECEIVED for review September 21,1981. Accepted November 5, 1981. The financial support of the Society for Analytical Chemists of Pittsburgh is gratefully acknowledged.
Electrospray Loading of Field Desorption Emitters and Desorption Chemical Ionization Probes Robert C. Murphy,* Kelth L. Clay, and W. Rodney Mathews Department of Pharmacology, Universltv of Colorado Medical School, 4200 East Ninth A venue, Denver, Colorado 80262
Mass spectrometry of complex, highly polar compounds typically encountered in biomedical research has been significantly advanced by the techniques of field desorption (FD) ( I ) and in-beam desorption techniques (2)such as desorption chemical ionization (DCI). Deposition of the sample onto the probe surface used in these techniques has remained as a difficulty especially when dealing with the fragile carbon microneedles of the FD emitters and only submicrogram quantities of materials. In the past, sample loading on such surfaces has been accomplished by the dipping technique (3) or microliter syringe transfer (4).Olson et al. (5) described an improved, quantitative technique for loading solutions by freezing the microliter droplets on the FD emitter wire. However, this method as well as the other procedures are limited to 1or 2 p L volumes which can be applied to a wire as well as being somewhat technically difficult and tedious to perform. McNeal et al. (6)recently described an electrospray method to deposit samples on foils for californium-252 plasma desorption mass spectrometric analysis. A relatively large surface area was covered with sample; however, it was suitable for thermally labile molecules, relatively dilute solutions, and solvent systems compatible with biologically derived substances. We have modified this electrospray technique for the loading of substances on FD emitters and DCI probes. The procedure is reproducible, rapid, highly efficient, and well suited for the poorly wetable and fragile carbon microneedles of the FD emitter.
EXPERIMENTAL SECTION Apparatus. The electrospray system is simple in design, consisting of a Hamilton 701N 10-pL syringe with a 26 gauge side port needle 5 cm long held vertically in a electrically isolated Plexiglass frame. Figure 1 is a photomicrograph of the needle tip. Positive high voltage is applied to the needle by a Power Design Inc., Model 1543A (0-10 kV, 10 mA) supply through a
50-MQcurrent limiter resistor. The sample probe to be loaded is mounted on a laboratory jack to position the emitter wire below the needle (2-10 mm) and is subsequently connected to an adequate electrical ground. The electrospray plume can be visualized by intense back lighting and the process monitored by inspection of the miniscus movement within the barrel of the syringe. Reagents. Solvents were distilled in glass grade obtained from Burdick and Jackson Laboratories, Inc. Glutathione and 2deoxyguanosine were obtained from Sigma, Inc. The 6-(Nacetylcysteinyl)-5-hydroxyeicosatetraenoicacid was synthesized by condensation of N-acetylcysteine with leukotriene A4 in a minimum volume of methanol with 1% triethylamine. Leukotriene A4 was a kind gift from J. Rokach, Merck-Frosst, Inc. Procedure. The solution to be sprayed is first loaded into the syringe and syringe placed in the electrospay apparatus. The plunger is then removed and the solution allowed to flow into the needle by either gravity or slight air pressure. It is important to avoid any air gaps in the liquid column since the electrospray will terminate when this gap reaches the needle tip. The syringe needle is adjusted to within 2-5 mm of the emitter wire, and high voltage is applied (typically 4 kV). The spray immediately begins as seen in Figure 2. If more than 10 pL is to be applied, the syringe may be reloaded as above or a resevoir attached to the syringe barrel. Estimation of transfer efficiency was carried out by using [35S]cysteinedissolved in methanol at 5.25 X lo5 (counts/ min)/mL. Five-microliter aliquots (2625 counts/min) were sprayed on the target wire, and the wire was carefully washed with methanol which was collected in a scintillation vial. Scintillation cocktail (Budget-Solve, Research Products International) was added, and the washings were subjected to liquid scintillation counting (Beckman LS-100). Field desorption and DCI were carried out on a Model 7070H double focusing mass spectrometer (VG Micromass). The desorption chemical ionization experiments were carried out using ammonia as reagent gas.
RESULTS AND DISCUSSION Needle-Tip Shape. The shape of the syringe needle was found to be critical for optimal transfer of sample to emitter wire. The best results of focusing the electrospray on the wire
0003-2700/82/0354-0336$01.25/00 1982 American Chemical Society
ANALMICAL CMMISTRY. VOL. 54, NO. 2. FEBRUARY 1982
337
Table 1. Effect of Applied Voltage and Electrode S p a ration on Transfer of [ "SI Cysteine by Electrospray" voltdisrecovage, tance,b ery,% kV mm (std error)= comments 4 4 4 4 3 5 6
2 5 10 15 5 5 5
99 (1.4) 95 (4.0) 70 (3.3) 56 30 (1.5) 93 (6.8) 83 (0.9)
escellent excellent wide plume poor
very poor excellent wide plume
DCI probe, electrospray 5 pL, 2625 countslmin total. *Distance from probe to needle. E n = 3, standard of the mean. Flpm 1. Photomicrogaph (14x1 of si& port need& used in e b bospray,scaieddMslons lmn: i A l n a d e l b * h c h e m M e d e x c e * n ( elecbo~pay8Ben in Fbue 2: (61 needle I p which had very p o a
ekbospray characleristlcs.
Table 11. Effect of Solvent System on Electrowray' solvent rate, % waterb pL/min 0
4.6
10 30
3.3 2.0 1.7
40
1.6
20
spray,' charaeistia
comments
++ + ++ +
some sputtering droplets formed on wire 50 1.4 0 sprayed only at 10 mm a 4 kV applied potential, 5 mm separation of needle point and emitter wire. In methanol, vlv. e + +, excellent: +. eood: 0. wor.
-
Flpm 2. Detail of me elecbospay of memanoilwater (8/2. vlv) onto a FDmrtlterwlre. Thed$tanca lmtween needle tip and W i n 5 mn. appikd potential 4 kV.
were obtained with rounded, sideport needle tips as Been in Figure 1A. I t was necessary to increase the voltage to 6 kV in order to obtain an electrospray from the sharp. side-port needle Been in Fibure 1B.At these higher potentials the spray from the sharp needle WBS very wide and poorly focused. This needle was rounded with emery cloth and then had electrospray characteristics identical with that of the needle in Figure 1A. With these rounded tips the electrospray plume characteristically had a narrow region extending 0.5-1 mm from the tip which spread out and focused on the grounded emitter wire as seen in Figure 2. As described by McNeal et al. (6). the solution is dispersed in this plume into sub-mimn-sized charged droplets which partially evaporate as they travel to the emitter wire. Solvent System and Electric Potential Gradient. The transfer efficiency of [%]cysteine in methanol by electrospray onto the DCI probe is summarized in Table I. The best results were obtained a t 2 mm separation of wire and needle tip with 4 kV applied potential. In general, the efficiency increased with decreasing Separation. Increasing the potential above 4 kV a t a 5 mm separation caused the spray plume to spread out and hecome focused more on the emitter posts.
0
Flpm 3. Mass specbun of lsukotrlsne E4 derfvsHve (WNacWcysleinyl)-shyaoxyekosatelraem24c acid dimethyl ester) obtained by DCI (NH,) and loaded by eleehospray.
Increasing the water content resulted in a parallel decrease in electrospray rate (Table 11). However above 30% water. droplets began to collect on the needle tip during eleetmspray which sputtered often to other grounded portions of the a p paratus. Very poor spray characteristics were ohserved with methanol containing 50% water. Mass Spectrometry of Thermally Labile Moleeules. The DCI mass spectrum of glutathione (-&lu-Cys-Gly) losded by electrospray has been compared to that loaded directly by microliter springe. Qualitatively the spectra were identical for this tripeptide which is easily oxidized and thermally unstable. Measurements of the abundance of the MH+ ion a t m/z 308 from both techniques were not statistically different when 0.33-1.33 pg were loaded. One additional advantage of the electrospray loading technique is that relatively large volumes of dilute solutions can he conveniently used thus eliminating the need to concentrate and work with microliter volumes. When pure methanol is used as the solvent, volumes of 100-200 rL can be conveniently loaded on DCI wires. Figure 3 is the DCI (NHJ spectrum of the thermally sensitive dimethyl ester of 6(N-aoetyleysteinyl)-5hydro~eicosatetrcacid obtained by eleetmsprayof 30 p L of a 0.13 mM solution. The important ions a t high mass characterintic of the intact molecule, MH+,
338
Anal. Chem. 19a2. 54, 338-339
Flgure 4. Flekl desorptlon mass spectrum of 2deoxyguanosine loaded onto the FD emltter wlre by electrospray in rnethanokwater (8:2).
loss of methanol, and loss of water are not observed if the sample is heated above room temperature during the loading process. Figure 4 is the field desorption mass spectrum of 2deoxyguanosine which was loaded on the FD emitter by electrospray. In addition to the molecular and cationized species at m / z 268, 290, and 306, strong ion currents were observed at masses corresponding to dimerized species: 2M H+, 2M Na+, and 2M K+.Considering the difficulties that can be encountered in obtaining the FD spectra of this compound, the electrospray procedure does not damage the FD emitter needles. The exact origin of the ions at m / z 441 and 373 is currently unknown.
+
+
+
CONCLUSIONS The method of electrospray loading of DCI probes and FD emitter wires has been demonstrated to be a useful technique for highly efficient transfer of sample. The electrospray apparatus is extremely simple to construct and operate. Elec-
trospray is a rapid and gentle technique which causes no damage to the very delicate carbon microneedles of the high temperature activated FD wires. Furthermore, the surface which is exposed to the electrospray plume corresponds to the surface juxtaposed to the extractor plate in the FD ion source and from which ions are desorbed. Thermally sensitive and easily oxidized samples can be loaded with this technique making this approach readily applicable to the analysis of most biological molecules by DCI and FD mass spectrometry.
ACKNOWLEDGMENT The authors thank J. Rokach, Merck Frosst Laboratories for the synthetic methyl ester of leukotriene A4. LITERATURE CITED (1) Reynolds, W. D. Anal. Chem. 1979, 51, 283 A-293 A. (2) Cotter, R. J. Anal. Chem. 1979, 51, 1589 A-1606 A. (3) Beckey, H. D. Int. J . Mass Spectrom. Ion Phys. 1969, 2 , 500-503. (4) Beckey, H. D. Hendrichs, A,; Wlnkler, H. Int. J. Mass Spectrom. Ion Phys. 1970, 3, App. 9. (5) Olson, K. L.; Cook, J. C.; Rinehart, K. L. Biomed. Mass Spectrom. 1974, 7 , 358-362. (8) McNeal, C. J.; Macfarlane, R. D.; Thurston, E. L. Anal. Chem. 1979, 57,2036-2039.
RECEIVED for review August 25,1981. Accepted October 26, 1981. This work was supported through grants from the National Institues of Health (HL25785 and RR001152). Presented in part at the Annual Conference of the American Society for Mass Spectrometry, Minneapolis, MN, 1981.
Demountable Holder for Modlfied Electrodes Brigitte Bolsseiler-Cocollos, Etlenne Lavlron,* and Roger Gullard" Laboratoire de Synthke et d'Electrosynth8se Organom6talllque assocl6 au C.N.R.S., LA 33, 6, Boulevard Gabriel, 21 100 Djion, France
Redox modified electrodes have recently been the object of much interest (1-4).Use of these electrodes for examining catalysis or charge transfer reactions requires durable attachment of the molecules containing the redox centers to the surface of the electrode. One of the best means of achieving this result consists in creating functional groups on the surface of an electrode and in using them to form a covalent bond with the molecules via an appropriate reaction (5-26). In some of the procedures which have been described for the functionalization (5-16), the electrode is subjected to rather drastic conditions (for example, in the case of carbon electrode, oxidation by O2a t 450 "C (5-12)) or plasma etching (7, 14), followed by reaction with SOClzin boiling toluene). Ordinary disk electrodes in which a cylinder of the electrode material is set in a tubing of a nonconducting material (glass, Teflon, -) do not withstand such treatments. This difficulty can be circumvented by treating a piece (usually a rod or a disk) of the material alone and by inserting it afterward into an appropriate holder. Murray et al. have used for example a heat shrinkable Teflon tubing to protect the cylindrical part of the carbon rod from the solution (7, 8, 9, 10, 13). We describe here an electrode holder which allows a rod of the electrode material to be mounted or demounted rapidly and which we think can be of service to electrochemists working in this field. It can also eventually be used as a rotating disk electrode. Holders of a different conception, in which carbon disks (11, 27) or cones (28) can be mounted have been described. EXPERIMENTAL SECTION Materials. The glassy carbon V25 was obtained from "Carbone Lorraine" (Paris, France) in the form of cylindrical rods which 0003-2700/82/0354-0338$01.25/0
were cut into pieces 4-5 mm in length. The poly(tetrafluor0ethylene) (PTFE) Gaflon was provided by Plastic Omnium (Sirem Division, Langres, France). Tetrakis (p-aminophenyl)porphyrin, TpNH2PPH2,was prepared by reduction of tetrakisb-nitropheny1)porphyrin according to the procedure described by Collman et al. (29) for the preparation of tetrakis(rn-aminopheny1)porphyrin. Dimethyl sulfoxide was freshly distilled under argon on active alumina. Tetraethylammonium perchlorate was dried in vacuo in the presence of PzOs. Apparatus. Current-potential curves were obtained by means of a commercial potentiostat (Tawse1PRT 30-0.1,Lyon, France) and recorded on an XY recorder (Sefram TGM 101, Paris, France). A one-compartment cell was employed with a platinum wire auxiliary electrode. The saturated calomel reference electrode was separated from the main cell compartment by immersion in a glass tube terminated by sintered glass frit.
RESULTS AND DISCUSSION The holder, which consists of four parts in PTFE, is shown unassembled and assembled in Figure 1. The rod electrode is inserted from above part A into the cylindrical part (a) where it fits exactly. This ensures in particular that the modified part, which will be exposed to the solution, is not handled during this procedure. Part B is then screwed on part A, which, owing to its conical shape, ensures that a tight contact is made between the rod and the holder. A glass or a stainless steel tubing is then inserted in the upper part of the holder. Air tightness is provided by the biconical joint C, by screwing part D on the top of part A; this ensures in particular that oxygen which is present inside the holder (in F) cannot leak into the electrochemical cell. With, e.g., a carbon or a platinum electrode, electrical contact can be made 0 1982 Amerlcan Chemlcal Soclety
