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circumvent some of these difficulties. We are in the process of developing such a system.
ACKNOWLEDGMENT The authors thank John Sadler (VG Analytical) for design of the MID circuitry, Ben Johnson (Finnigan-MAT) for modifications to the interface to the data system, and Mark Weiss (Finnigan-MAT) for helpful software discussions.
LITERATURE CITED (1) Beynon, J. H. “Mass Spectrometry and Its Application to Organic Chemistry”; Eisevier; Amsterdam, 1960;pp 312. (2) Kimble, B. J. I n “High Performance Mass Spectrometry: Chemical Applications”; Gross, M. L., Ed.; American Chemical Society: Washington, DC, 1978;Chapter 7; ACS Symp. Ser. No. 70. (3) Hadden, W. F.; Lukens, H. C. Presented at the 22nd Annual Confer-
ence on Mass Spectrometry and Allied Topics, Philadelphia, PA, 1974,
u-2.
(4) Craig, R. D.; Baternan, R. H.; Green, B. N.; Millington. D. S. Ph//os. Trans. R. Soc. London, Ser. A 1079, 283, 135-155. (5) Hammar, C. G.; Hessiing, R. Anal. Chem. 1071, 43, 298-306. (8) Hammar, C. G.; Pettersson, G.; Carpenter, P. T. B/omed. Mass Specfrom. 1074, 1, 397-411. (7) Seller, N; Knoedgen, B. Org. Mass Spectrom. 1973, 7. 97-105. (8) Meredith, J. 0.; Southon, F. C.; Barber, R. C.; Williams, P.; Duckworth. H. E. Int. J . Mass Spectrom. Ion Phys. 1073, 10, 359-370. (9) Gruenke, L. D.; Cymerman, C. J.; Bier, D. M. B/omed. Mass Spectrom. 1074, I, 418-422. (10) Hadden, W. F. I n “High Performance Mass Spectrometry: Chemlcai Applications”; Gross, M. L., Ed.; American Chemical Society: Washington, DC, 1978,Chapter 6;ACS Symp. Ser. No. 70.
RECEIVED for review August 31, 1981. Accepted October 23, 1981.
Microelectrolytic Cell for Voltammetric Analysis Joseph Wang’ and Bassam A. Freiha Department of Chemistty, New Mexico State UniversiW, Las Cruces, New Mexico 88003
It has been demonstrated in the literature that electroanalytical techniques can be adapted to analyses of very small sample volumes. Most electrochemical cells with small volume capability are based on the thin-layer principle (usually in a “sandwich type” configuration) ( I , 2). Other cells or approaches include a rotating disk electrode microcell (3, 4 ) , capillary packed-bed electrodes (5),mercury drop electrode microcells (6),specially designed cells with stationary carbon electrode (9, as well as micro flow cells for liquid chromatography and flow injection analysis (8). Thin-layer cells suffer from problems such as “iR distortion” or “edge effect” (9, IO), and they are relatively expensive and difficult to construct. With the growing need for sensitive and reliable analyses in microliter sample volumes it becomes necessary to explore other microcell configurations. We report here a very simple and inexpensive microelectrolytic cell. The cell utilizes a drop of the test solution placed on an inverted carbon paste disk electrode, with a capillary reference electrode immersed in the drop. The sample solution defines its geometry over the electrode via surface tension. A cell of similar philosophy, but of different geometry, has been described (7). However, little has been reported concerning the response characteristics of this cell. Advantageous features of the present cell include simplicity and low cost of construction, ease of use, and sensitive and reproducible response. Thus the cell minimizes some of the disadvantages of thin-layer cells. Measurements are performed on 50 p L solution volume. Differential pulse voltammetry (DPV) is employed for measuring dopamine, chlorpromazine, and ferrocyanide ion at the micromolar concentration level. EXPERIMENTAL SECTION The cell design is shown in Figure 1. The body consisted of two 3.5 X 3.0 X 1.2 cm Plexiglas blocks, held together with two stainless steel bolts (at opposite corners). A 0.3 cm diameter hole was drilled in the center of the lower block to accommodate the carbon paste working electrode. The paste was made by thoroughly mixing graphite powder (Acheson Graphite, Grade 38, Fisher) and Dow Corning silicone grease (43% silicone grease by weight). The paste surface was smoothed using a computer card, and ita other end was connected to a copper wire (epoxied in the hole) that led to the outside. The geometric area of the working electrode is approximately 0.07 cm2. A 50-pL sample drop was placed on the carbon disk, covering all the surface of the electrode. A hole (0.5 cm widened to 1.5 cm diameter) was drilled in the center of the upper block to contain the reference electrode and protect the sample against contamination and evaporation effects
(such protection was not provided in the cell described in ref 7). The Ag/AgCl reference electrode was made of a capillary tube (Kimble Products, Toledo, OH) of 1.8 mm o.d., which contained a thin silver wire and 0.1 M KC1 solution. The reference electrode was immersed in the solution drop, about 2 mm above the center of the carbon paste disk (the close proximity of the two electrodes minimized distortion effects due to ohmic polarization). Samples were removed by washing the electrodes with deionized water, followed by drying the working electrode with a stream of air. A 50-pL Hamilton microsyringe was used to place new samples. In all experiments, a Princeton Applied Research Model 364 polarographic analyzer was used with a Houston Omniscribe chart recorder. Chemicals and reagents used have been described previously (11) excepted as noted. Chlorpromazine hydrochloride was obtained from Sigma Chemical Co. All solutions were prepared daily by weight in 0.1 M phosphate buffer, pH 7.4.
RESULTS AND DISCUSSION Figure 2 shows linear scan and differential pulse voltammorgrams for the oxidation of 10 FM dopamine and 100 HM chlorpromazine in pH 7.4 phosphate buffer. The voltammograms are well-defined (and reproducible) and the background current is low. The DPV peak potentials for dopamine and chlorpromazine are a t +0.08 and +0.8 V, with widths a t half-height of 93 and 210 mV, respectively. Voltammograms with similar potential regions and shapes have been observed for dopamine and chlorpromazine at various carbon electrodes (12,13). The dopamine linear scan response exhibits a nearly steady-state plateau rather than diffusion limited current decay. This behavior may be attributed to radial diffusion to the edges of the disk, as will be discussed later in the paper. The advantages of DPV, over linear scan voltammetry, for determining low concentrations of easily oxidized organic molecules are obvious. Detection limits at the submicromolar concentration level are predicted from the signal to noise characteristics of the DPV data. The degree of carry-over, i.e., the influence of the concentration of one solution on the result obtained for the subsequent solution, may be tested by sequential determinations of sample and blank solutions. Results of such a test run with 50 pM dopamine solution followed by ita corresponding blank (0.1 M phosphate buffer) solution are shown in Figure 3. Well-defied peaks (with similar peak heights-4.99,0.98, and 1.01 pA) are obtained, with no observable carry-over between the sample and blank solutions. The sample replacement procedure (washing, drying, etc.), described in the Experi-
0003-2700/82/0354-0334$01.25/00 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982
C-
1
I
1cm
335
P
,
_i
I
B
-F=-----
L-
Figure 1. Microcell for voltammetric measurements: (A) sample solution; (B) carbon paste working electrode; (C) capillary silver-silver chloride reference electrode: (D) copper lead to working electrode.
IC
A
.2
0.2
0.6
Flgure 3. Carry-over between sequentlal blank (B) and sample (S) solutions: (S) 50 pM dopamine in 0.1 M phosphate buffer, (B) 0.1 M phosphate buffer: hear scan voltammetry; scan rate, 50 mV/s.
.-
0.1 0.5 0.9 E ,v Figure 2. Electrochemical oxidation of 10 pM dopamine (A, B) and 100 pM chlorpromazlne (C, D) in 0.1 M phosphate buffer, pH 7.4. Conditions: Dlfferentlal pulse voltammetry (A, C), scan rate, 5 mV/s; amplitude, 50 mV. Linear scan voltammetry (B, D), scan rate, 50 mV/s. Dotted lines represent blank solution. -0.2
0.2
0.6
mental Section, is essential for minimizing carry-over effects. Under these conditions a new sample may be measured within 1min. Thus with linear scan voltammetry and DPV used in the measurement step, sampling rates are about 60 and 18 samples per hour, respectively. Further improvement of the sampling rates for measurements at the micromolar concentration level may be affected by employing rapid scan potential pulse procedures (14, 15), not available yet in commercial instruments. Quantitative evaluation of the cell is based on the linear correlation between the peak current and the analyte concentration. Linear scan voltammograms for sequential ferrocyanide samples of ascending concentration (0.2-1.0 mM) are shown in Figure 4. The resulting plot of peak current against concentration (not shown) is highly linear with a slope of 7.23 pA/mM. Thus, rapid quantitation can be effected by comparing the peak height for the unknown concentration with that of a standard solution. The linear correlation found again showed carry-over effects to be negligible. The reproducibility of results is indicated by ten repetitive determinations of 0.5 mM ferrocyanide, using the same sample drop (conditionsas in Figure 3). The mean current difference found was 2.73 pA with a range of 2.70-2.81 pA. The relative standard deviation (RSD)over the complete series was 1.4%. A slight decrease in precision (RSD of about 5 % ) was observed by using DPV; this may be attributed to the longer time scale
-0.2
0.2 0.6
6v
Figure 4. Linear scan voltammograms for sequential ferrocyanide solutions of ascehding concentration in 0.1 M phosphate buffer: scan rate, 50 mV/s. of the DPV experiment during which more alterations (e.g., depletion or evaporation effects) can take place. As long as bulk depletion of the analyte is negligible (i.e., rapid scan experiments) all the principles and equations of faradaic electrochemistry at a planar disk electrode in a quiescent solution (16) can be applied to the cell. Since no forced convection is being employed, diffusion (perpendicular as well as radial to the edges of the disk surface) serves as the only mode of mass transport. When a smaller working elec-
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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-pLsyringe 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 (typically4 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
