Anal. Chern. 1980, 52,377-379
377
electrodes with no change in equipment. For example, Figure 4 shows a standardization curve for an Orion chloride electrode used in the inverted mode with a reference microelectrode as in Figure 1.
LITERATURE CITED
1
2
3
4
P [CII
Figure 4. Electrode response to chloikle concentration using the chloride
electrode assembly described in the text samples should be run in duplicate or triplicate. Such contamination can usually be eliminated by reconditioning the electrode surface, or recleaning the micropipets. Carryover from the reference microelectrode is rarely a problem, but it can be eliminated entirely by passing the tip of this electrode through a small drop of distilled water on the electrode surface before bringing it into contact with the next specimen. By comparison to other methods (1-3) of modifying solid state electrodes for use with microsamples, the method described here has numerous advantages; it is faster, uses much smaller volumes, and can be used with most solid state
(1) A. S.Hallsworth, J. A. Weatherell, and D. Deutsch, Anal. Chem., 48, 1660 (1976). (2) D. H.Retief,'J. M. Navia, and H. Lopez, Arch. OralBiOl., 22, 207 (1977). (3) A. Venkateswarlu, Anal. Chem., 46, 878 (1974). (4) L. A. Geddes "Electrodes and the Measurement of Bioelectric Events", Wiley-Interscience, New York, 1972. (5) "Physical Techniques in Biological Research", W. L. Nastuk, Ed., Academic Press, New York, 1972. (6) D. J. Prapr, R. L. Bowman, and G. G. Vurek, Science, 147, 606 (1965). (7) H. 0.Lowry, N. R. Roberts, K. Y. Leiner, M. Wu, and A. Farr, J. Biol. Chem., 207, l(1954).
RECEIVED for review June 6,1978. Resubmitted July 31,1979. Accepted November 2,1979. This investigation was supported in part by Research Grants DE04385 and RR05689 to the American Dental Association Health Foundation from the National Institute of Dental Research and is part of the dental research program conducted by the National Bureau of Standards in cooperation with the American Dental Association Health Foundation. Certain commercial materials and equipment are identified in this paper in order to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the material or equipment identified is necessarily the best available for this purpose.
Microanalytical Techniques with Inverted Solid State Ion-Selective Electrodes. 11. Microliter Volumes Gerald L. Vogel" and W. E. Brown American Dental Association Health Foundation Research Unit, National Bureau of Standards, Washington, D.C. 20234
We reported recently ( I ) how a commercial solid state electrode may be adapted for microvolumes by bringing a glass microreference electrode into contact with hemispherical microdrops of specimen deposited under mineral oil on the surface of the electrode mounted in an inverted position. While this method is fast (20 to 30 specimens per hour), and although specimens of microscopic size can be determined (300 pL and less), it is somewhat elaborate for routine laboratory use with specimens of 1 to 5 FL. In this paper we describe a simple device that will, in a few minutes, adapt most solid state electrodes for the rapid determination of samples in this volume range.
EXPERIMENTAL An Orion fluoride electrode (94-09-00) is adapted for use in the
inverted position by filling it carefully with fluoride reference solution so as to exclude air bubbles. After filling, a thin coat of hot dental wax is applied to the sensing element, and the electrode is clamped in an inverted position. The adapter consists of a nylon cylinder, approximately 12.5 mm in diameter by 5 mm, in which seven 2-mm holes have been drilled (Figure 1). The device is affixed to the sensing element of a solid-state electrode by gently heating it in an oven or on a low-temperature hot plate and pressing it onto the wax-covered electrode. A 2-mm drill bit is heated and pressed blunt end first into the holes in the adapter, and any hot melted wax forced out by this procedure is removed with a tissue. The remaining wax in the holes is carefully removed with a small piece of cotton
wrapped around a toothpick and moistened slightly with chloroform. Gentle removal of the wax is essential to prevent leaks between the wells. A reference electrode for use with the adapter is constructed as follows (Figure 1): (1)Heavy wall borosilicate glass tubing, 8mm 0.d. by 1.3-mm i.d., is pulled into a thin capillary, -0.15-mm id.; a section of this capillary is then pulled to a still smaller size, -0.03-mm inner diameter; and this section is cut with a diamond or carbide scribe to give a capillary of the shape shown in Figure 1. (2) The capillary is inserted into a short section of fine plastic tubing, -1.0-mm i.d., connected to a tapered glass tube attached to the bottom of a small two-neck distillation flask. One neck of this flask contains a rubber stopper through which is inserted a calomel reference electrode connected to the reference input of the electrometer. (3) The capillary tubing and the two-neck flask are filled with a solution of filtered 0.2% agar containing 1M KC1. The agar will prevent the KC1 solution from emptying out of the electrode by gravity. If the agar is too stiff or the capillary too fine, the agar may tend to pull away from the tip of the capillary and thus break electrical contact with the solution. This will result in unstable reading. Raising the two-neck flask relative to the adapter or applying pressure on a syringe inserted through a rubber stopper in one neck of the flask should stabilize the readings immediately. If it does not, the problem is probably not associated with the agar but is caused by a blocked capillary. This can be replaced easily without affecting the measured values. Fluoride standard buffers containing 50% Orion Total Ionic Strength Activity Buffer are deposited in the holes of the adapter with a Pipetman model p-20 pipetter (Rainin Instrument Co.,
This article not subject to U S . Copyright. Published 1980 by the American Chemical Society
378
ANALYTICAL CHEMISTRY, VOL. 52, NO. 2, FEBRUARY 1980 Rubber Stopper
*O0 Electrometer
2 Neck Flask
c
'
,,Glass
F C
tt
/HOLE
7
- .2% Agar. 1 m KCI Tube
I /
A UPRIGHT ELECTRODE
i o ml
I
Tygon Tubing
10
1
I
SAMPLES 1
10 Fluoride Concetrbation (molesAiterl 10-4
I 10
Figure 2. ElecWode adapter performance with an Orion fluoride electrode (3-pL samples)
Plastic - -Slide Cover (with hole) Orion Fluoride Electrode
,'
uMini Sample Adapter
Figure 1. Microliter adapter and reference electrode
Brighton, Mass.). The dimensions of the holes in the adapter and its thickness were chosen so as to be compatible with the dimensions of the disposable tips used with this pipet. Measurement Procedure. Absorbed fluoride within the wells is a potential source of contamination. This problem may be avoided by conditioning each hole of the adapter between samples as follows: Each hole of the adapter is washed with p[F] 6 standard using a hypodermic syringe. p[F] refers to the negative log of the fluoride concentration (moles per liter). The conditioning solution is allowed to remain in the holes of the adapter for 10 min before new specimens are introduced. Conditioning fluid or specimens can be throroughly removed from the holes of the adapter with a section of fine plastic tubing attached t o a vacuum source. After the samples are deposited in the adapter, a small plastic microscope cover slide with a 1.5-mmhole is placed over the top to prevent evaporation, and the capillary tip of the reference electrode is inserted into a specimen through this hole (Figure 1). After the measurement, the capillary is removed from the hole, rinsed with distilled water, and inserted into the next specimen. Calibration is easily accomplished by placing a different standard in each hole of the adapter (Figure 2). This procedure will also immediately detect any leaks between the wells since leaking wells will show readings that are obviously incorrect by comparison to the other standards.
RESULTS AND DISCUSSION Figure 2 presents two standardization curves for the fluoride electrode. In the upper curve, seven 3-pL samples ranging in concentration from p[F] 3 to p[F] 6 were measured in each hole. The standard deviation between the holes was 0.4 mV a t each level except p[F] 5.3 where it rose to 0.7 mV and p[F] 6 where the standard deviation was 1.7 mV. The standard deviation from linear regression between p[F] 3 and p[F] 5, (the linear range), was 0.4 mV for each hole, implying an accuracy of 0.007 p[F] unit. By repeatedly inserting the reference electrode alternately in p[F] 3 and p[F] 6 samples in adjacent holes, the carryover of the reference was tested and found not significant. Since conditioning was shown to be very important in microvolume measurements with standard electrodes ( 2 ) ,several conditioning solutions were tested to determine which one was most
effective in removing p[F] 3 solution from the wells prior to insertion of 3 pL of p[F] 5 standard. Conditioning with p[F] 6 was very effective, requiring only 7-10 min to condition the wells so that the 3 pL p[F] 5 sample held a constant reading within 1 mV of the known correct value. When the conditioning solution was p[F] 5 (the same concentration as the test solution), it was found to be a relatively ineffective agent requiring equilibrium times in excess of 20 min to effectively condition the wells. p[F] 6 conditioning has the advantage over distilled water of providing a well-defined millivolt reading to compare with in order to determine when the process is complete. Usually 90% of the correct p[F] 6 values is sufficient unless samples more dilute than p[F] 5.3 are anticipated. Samples which are expected to be very similar (such as dental enamel biopsy specimens) may require little or no conditioning if highest accuracy is not sought. If any samples are found to be dissimilar, they can be run later. Compared to other methods of adapting standard electrodes to minivolumes (2-5), the adapter has several advantages including smaller sample size and faster determinations. The speed of this method is primarily a result of the fact that, since all seven samples can be deposited a t nearly the same time, they all approach equilibrium concomitantly, Furthermore, the same type of adapter can be used with most solid-state electrodes. Recently, by replacing the KC1 solution with KNOB,Popp, Frantz, and Vogel (6) have used the same type of adapter with a chloride solid-state electrode. Finally, because of the high accuracy of this method, it can be used in place of the usual fluoride electrode procedure with considerable savings in both time and equipment since this system replaces seven solid-state electrodes, their reference electrodes, and a switching mechanism. The lower curve in Figure 2 shows the same fluoride electrode with macrosample volumes and a calomel reference electrode in the usual (clamped upright) mode. Ten-mL samples were used and a 50-mV offset was applied to separate the two curves. The equivalence of the two techniques can be seen since the two curves are essentially parallel, except in the p[F] 5 to 6 range, where the upper curve, (microvolumes), falls off slightly more rapidly. LITERATURE CITED (1) G. L. Vogel, L. C. Chow, and W. E. Brown, Anal. Chem., preceding paper in this issue. (2) A. S. Hallsworth, J. A. Weatherell, and D. Deutsch, Anal. Chern., 48, 1660 (1976). (3) D. H. Retif, J. M. Navia, and H. Lopez, Arch. OralBiol., 22, 207 (1977). (4) R. A. Durst and J. K. Taylor, Anal. Chsm., 39, 1374 (1967). (5) R. A . Durst and J. K. Taylor, Anal. Chern., 39, 1483 (1967). (6) R. K. Popp, J. D. Frantz, and G. L. Vogel, In press.
RECE~VED for review July 24,1978. Resubmitted July 31,1979. Accepted November 2,1979. This investigation was supported in part by research grant No. DE04385 to the American Dental
Anal. Chem. 1980, 52, 379-384
Association from the National Institute of Dental Research and is part of the dental research program conducted by the National Bureau of Standards in cooperation with the American Dental Association Health Foundation. Certain commercial materials and equipment are identified in this paper
379
in order to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the material or equipment identified is necessarily the best available for this purpose.
Microcomputer-Assisted Mass Spectral Data Acquisition System Utilizing a Mass Marker Brian D. Andresen,”‘ William A. Wagner, and Frank T. Davis University of Florida, College of Pharmacy, Gainesville, Florida 326 10
Computer-assisted mass spectral data acquisition has achieved significant advances in the past decade. Through the use of large-to-medium computers with sufficient memory capacity, speed, and appropriate programs, low resolution mass spectral data can now be acquired, digitized, stored, and manipulated most easily (1-5). I t has now become essential for most applications that a computer control all aspects of the mass spectral data gathering. This would include continuous scanning of the mass spectrometer during any gas chromatographic-mass spectrometric (GC-MS) analyses of complex mixtures, acquisition of the data from the instrument, the processing of the data, and orderly arrangement of the results for rapid human interpretation. The favorable capabilities of a computer-assisted mass spectral data acquisition system would allow larger volumes of data to be acquired and processed, faster data access for interpretation, and permanent data storage. As a further extension, the interpretation of mass spectral data can be left completely under the control of the data system which accurately searches known library compilations for the nearest spectral match (6-16). In this manner, complex mixtures can be completely characterized without any human interpretation in the shortest possible time (6). Investigations into the availability of commercial computers, programmed for mass spectrometry data acquisition, demonstrated that most were relatively expensive. Complete GC-MS-computer systems could be obtained for $50 000$60 000 that possessed dedicated programs for mass spectral data acquisition and display. However, these systems were considered to be too costly and, in addition to duplicating existing analytical equipment, they could not easily be interfaced to another mass spectrometer. Many mass spectrometry research centers have indeed successfully interfaced rather large and sophisticated computers to their own instruments. However, these complex and refined data systems have been developed over a period of many years and are difficult to achieve and nearly impossible to duplicate. Recent developments in the field of microcomputer technology appeared to contribute all aspects of the much larger systems (i.e., speed and calculating power), yet were very cost effective if applied to mass spectral data acquisition only. Current literature has revealed only a limited number of microcomputer data systems which have been applied to an analytical task. These include automated titrations (17-19), a low-pass filter to smooth analog data (20),a microcomputer controlled dual wavelength controller (21), photoacoustic measurements using a microcomputer (22), as well as the commercial gas and high pressure liquid chromatographs. In these applications the microcomputer contributed significantly to the overall effectiveness of the analytical technique. In addition to these favorable reports, very recent developments in mass storage systems have become available. New “floppy ‘Present address and reprints: Department of Pharmacology,The Ohio State University, 333 West Tenth Ave., Columbus, Ohio 43210. 0003-2700/80/0352-0379$01 .OO/O
disk” storage systems could be interfaced to the microprocessor allowing large blocks of data to be stored and retrieved rapidly. This new technology seemed ideally suited for cost-effectively storing large blocks of data common to GC-MS runs and mass spectral libraries, which are often an integral part of normal mass spectral activities. All of these preliminary findings and unique features appeared most favorable for allowing a microcomputer to be interfaced to a mass spectrometer. A recent report has indicated that this approach is feasible with quadrupole mass analyzers (23, 24). The objective of the work reported here was to design a user-oriented GC-MS-microcomputer (GCMS-MC) system that could be applied easily to a DuPont 491 magnetic sector mass spectrometer. Many new microprocessors were currently available to accomplish the data acquisition, however, a Z-80-based microcomputer system was chosen because of its versatile and large (158) machine code instruction set incorporating blockdata-transfer capabilities. This function appeared essential when moving a whole mass spectrum in memory with the fewest machine code statements in the shortest period of time. A variety of designs was attempted, utilizing different combinations of hardware and software which would be most amenable to continuously acquire, process, store, and display mass spectal data. A practical design was eventually developed which was not simply a controller, but a user-oriented data system capable of meeting the goals outlined above, and possessing the capabilities to expand both the hardware and software when more complex tasks are required. The primary goal was simply to utilize a microcomputer system to aid in the rapid analysis of drugs and metabolites. Mass spectral data were to be acquired in a digitized form and then compared to a limited library of compounds. However, once the system was implemented, the emphasis was to develop additional programs to more fully utilize the data that had been stored by the microprocessor. The completed data system could acquire data and search a library file of’ known compounds as well as present hardcopy data of the results. We report here how this was accomplished and in addition describe the automated analysis of drugs from the body fluids of overdose victims.
INSTRUMENTATION The microcomputer system interfaced to a DuPont 491, double-focusing magnetic sector mass spectrometer is shown schematically in Figure 1. The computer system was assembled from commercially available components (Digitrl Group, Denver, Colo. 80206) and operated at 2.5 MHz using an 8-bit word, 2-80 microprocessor (Zilog,Inc., Los Altos, Calif‘. 94022), 32K of random access memory (RAM, TMS 4044) (Exatron, Sunnyvale, Calif. 94086), two SA801 shugart disk drives and controller (Digital Group), and an input/output interface (Digital Group) coupled to an audiocassette deck (Wevcor Mer. Co.), television (Sanyo VM-4209, Compton, Calif. 90220), Diablo printer (Data Dimension, Inc., Greenwich, Conn. 06830), and keyboard (Digital 0 1980 American Chemical Society
