Microwave Excitation Emission Spectrometry. Determination of Picogram Quantities of Metals in Metalloenzymes Hiroshi Kawaguchi’ and Bert L. Vallee2 Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, and the Division of Medical Biology, Peter Bent Brigham Hospital, Boston, MA 021 15
Optimum operating conditions have been determined for the excitation of metals in a low-pressure, microwave-induced helium plasma using a tantalum filament vaporization system. Addition to the sample of 4 to lOmM potassium chloride enhances the spectral line intensity of many elements and eliminates or suppresses interference effects. Detection limits for 8 elements measured in 5-pl samples are in or below the ng/ml range, i.e., to lo-’’ g of metal on an absolute basis. The precision of the method is demonstrated by zinc determinations in 5-pl samples where the relative standard deviation is 4.4% for 10 ng/ml zinc, while accuracy was established by analysis of metalloenzymes of known zinc stoichiometry. The method was applied successfully to the determination of metals in the reverse transcriptase from avian myeloblastosis virus.
A number of recent reports have dealt with the analytical applications of low-wattage, microwave-induced inert gas plasmas serving as an emission source for spectroscopic excitation (1-12). Relatively few of these studies have utilized reduced pressure during excitation (10-12), possibly because of the complexity of the vacuum system required and the difficulty encountered in introducing the sample into the plasma. We have undertaken studies employing a recently developed microwave-induced emission spectrometer (13) which obviates certain of these problems. The instrument employs a low-pressure, microwave-induced helium discharge, the sample being pipetted onto a tantalum filament, dried, ashed, and then vaporized into the helium flow cell, An electronically controlled automatic system facilitates the operation of the instrument. Fundamental properties of a low-pressure, microwaveinduced argon plasma have been studied ( 1 4 ) and the advantages which accrue to emission spectrometric analysis owing to thermal atomization of samples from filaments have been discussed ( 1 5 ) . However, detailed studies combining filament vaporization of samples with excitation in a low-pressure, microwave-induced plasma are not on record. The present study explores the characteristics of the low-pressure helium discharge and evaluates its analytical suitability, special attention being directed to interactions of the analyte with extraneous ions and their potential effects on detection limits. EXPERIMENTAL Instrumentation. The instrument employed in this investigation was designed and constructed by Monsanto Research Corp., Dayton Laboratories, Dayton, OH. A block diagram of the overall apparatus is shown in Figure 1. The components of the instrument, consisting of a microwave generator, optical system, gas flow control, sequence control, and signal readout system, are mounted on a standard rack cahinet, 42 inches high and 24 inches deep.
Present address. Faculty of Engineering, Nagoya University, C$kusa-Ku, Nagoya, Japan. - Author to whom correspondence should he add-essed.
X I pump
1
D.C. Amplifier
I
I
Storage
I oscitlosiope
II
] ‘ Peak detector Peak inteardor
Digital printer
Figure 1. Block diagram of apparatus
Figure 2. Schematic diagram of the mounting of cavity and discharge tube (A) Teflon plug, (B) Tantalum filament, (C) Vaporization chamber, ( D ) Discharge tube, (E) Plasma, (F) Microwave cavity, (G)Quartz lens, (H) Mounting plate, (I) Vernier advance knob
T h e microwave generator (Monsanto Research Corp., Model 207) supplies maximum power of about 100 W a t 2450 MHz into a 50-ohm matched load. A schematic diagram of the discharge tube assembly is shown in Figure 2. An Evenson quarter-wave cavity, F (Opthos Instrument Co.), couples the power from the microwave power source to helium gas, which flows into a vertically positioned quartz discharge tube, D (3.2-mm i.d., 4.3-mm o.d., and 150 mm long) in which the helium plasma is generated. T h e cavity, the discharge tube, and the vaporization chamber, c, are mounted on a metal plate, H . T h e entire assembly can be moved vertically hy means of a vernier advance knob, I, so that any part of the discharge can be positioned to coincide with the optical axis of the entrance slit and monochromator. The cavity can be tuned easily to zero reverse power and, as long as the operating conditions remain constant, virtually no retuning is needed. T o maintain a constant flow of gas into the discharge tube, a two-stage pressure regulator, a single-stage pressure regulator, and a small bored orifice are placed between a helium tank and the discharge tube in this order. T h e flow rate of gas is controlled by adjusting the single-stage pressure regulator. A rotary vacuum pump (Welch, Model 1402) connected to the helium gas line maintains reduced pressure of 3-4 Torr in the discharge tube. A sample vaporization chamber, c, is located a t the top of the discharge tube and accommodates a removable Teflon plug, A, to which a sample loading filament, B, is attached. T h e 0.25-mm diameter tantalum wire filament is V-shaped, 14-mm high with a round bottom of 2-mm diameter. T h e current to heat t h e filament is produced hy a constant voltage dc source, with a maximum voltage of 15 V, and a high-capacity condenser of 0.216 ANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975
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04
1 30 50 DISTANCE FROM CAVITY CENTER, rnrn
IO
Figure 3. Effect of microwave power on the intensity distribution of Zn 2138.56-A along t h e discharge tube
intensity, arbitrary units, zinc concentration, 0.01 figlmi. Microwave power: ( 0 )30 w: (m) 40 w: (A)50 w
F. An optical pyrometer (Leeds and Northrup, Model 8620) serves to measure the temperature of the filament. On vaporization of the sample, it is introduced into the plasma and instantaneously emits radiation characteristic of the elements present. The radiation emitted is focused on the entrance slit of the monochromator by a 40-mm focal length quartz lens, G, 20-mm in diameter, attached to the monochromator in standard fashion. The monochromator (McPherson Instrument Corp., Model 218) employs two aspheric 0.3-m focal length mirrors and a 1200 lines/mm plane grating blazed a t 3000 A with a reciprocal linear dispersion of 26.5 A/mm. Both the entrance and exit slits are set a t a width of 50 pm and a height of 4 mm. A photomultiplier tube (EM1 9783B) is attached to the exit slit and is operated at an 850-V drop between anode and cathode. Hollow-cathode lamps serve as sources to precisely adjust the wavelength exiting from the monochromator. The photomultiplier tube is connected to a fast response dc amplifier followed by an electronic peak detector and a peak integrator. The very rapid (2-10 msec rise time) signal profiles cannot be recorded by means of an ordinary strip chart recorder. The peak detector serves the peak value of the signal and the integrator then measures the area under the curve, and these values are stored until printed on a digital printer (Newport, Model 800). The amplified signal is also monitored with a dual-trace, storage oscilloscope (Telequipment, DM64). The analytical sequence including desolvation, vacuum control, microwave output, sample vaporization, and display are programmed by an electronic controller. Reagents. Stock solutions were prepared by dissolving pure metals of reagent grade salts (Johnson, Matthey and Co., Fisher Scientific Co., Mallinckrodt Chemical Works, and Merck and Co.) in dilute acid or distilled water. Cadmium, cobalt, copper, lead, magnesium, manganese, and zinc were used as the nitrates and all other metals as the chlorides. Immediately before use, these stock solutions were diluted to appropriate concentrations with metalfree distilled water from a Pyrex still (Barnstead Co., 4 l./hour) preceded by a string filter (Commercial Filters Co., BRXB), a colloid-removing filter, and a mixed-bed, ion-exchange filter (Barnstead Co.). Ultra high purity grade helium (Union Carbide Co., 99.999%) served as the plasma gas. O p e r a t i n g Procedure. Samples of 5 pl are applied to the filament loop using an Eppendorf Microliter pipet. The filament is placed into the vaporization chamber through which helium flows at a rate of about 0.4 I./min. The solvent is vaporized slowly by passing a current of 2-3 A through the filament. This desolvation current is determined experimentally, the criteria being that the 5-pl sample solution should be evaporated to dryness in 30-40 sec and, further, that organic materials, when present, be decomposed during an additional 20-30 sec at the same current. On the one hand, when evaporation proceeds too rapidly, some of the sample may be lost by boiling; while, on the other hand, incomplete decomposition of organic material reflects as an irregular shaped peak in the oscillogram causing erroneous results. After proper desolvation is completed, both the vaporization chamber and the discharge tube are evacuated to a pressure of 3-4 Torr, a continuous helium flow being maintained. The microwave discharge, initiated with a Tesla coil, is allowed t o stabilize for about 20 sec before the sample is introduced. To vaporize the sample deposited on the filament, the high-capacity condenser, charged to an appropriate voltage, determined experimentally, is discharged through the filament, heating it to more than 1800 “C. Subsequent t o the discharge of the condenser, a dc current of 3.5 A is maintained for 2 sec to complete vaporization. 1030
ANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975
In the “automatic mode” of operation, all processes subsequent to deposition of the sample described above automatically proceed in sequence, and both the peak and integral values of special intensity are printed out digitally. For example, an analytical cycle was programmed experimentally to terminate in 103 sec (desolvation and ashing); 61 sec, evacuation: 10 sec, stabilization of discharge: 22 sec, sample vaporization: 2 sec, display and reset: 8 sec). Both maximal and integral intensities of a given signal are obtained, but since the precision of both is the same, the more convenient maximal intensity was chosen as a measure of concentration. While under normal operating conditions a filament can be used for 400 to 500 samples, the lifetime of the filaments tends to be shortened when the sample contains large amounts of organic materials of high molecular weight, e.g., protein.
RESULTS AND DISCUSSION Optimization of Discharge Tube Diameter. Q u a r t z t u b e s varying f r o m 1- t o 7 - m m i.d. were used to e s t a b l i s h the dependence of s t a b i l i t y of the p l a s m a and the emission i n t e n s i t y of metals o n t u b e d i a m e t e r . If the i n t e r n a l d i a m e t e r is less than 2 m m (at a power of 30 W), t h e p l a s m a is unstable; but stability increases as t h e d i a m e t e r increases. As t h e i n t e r n a l d i a m e t e r increases f r o m 3 t o 7 m m (at 30 W ) , t h e l e n g t h of the p l a s m a also increases f r o m 6 to 8 c m , b u t , simultaneously, the l e n g t h of t h e p l a s m a also increases f r o m 6 to 8 c m , b u t , simultaneously, the signal decreases. A t u b e of 3.2-mm i.d. a n d 4.3-mm 0.d. w a s a d o p t e d as o p t i mal and used t h r o u g h o u t t h i s investigation. W i t h respect to c o n t a m i n a t i o n , deposition of s a m p l e v a p o r o n the wall of t h e discharge t u b e is n o t a serious p r o b l e m since s e q u e n t i a l d e t e r m i n a t i o n s give no evidence of a “ m e m o r y effect”, i.e., t h e r e was no emission f r o m m e t a l s d e p o s i t e d o n the wall. However, with increasing n u m b e r s of d e t e r m i n a t i o n s , a w h i t e d e p o s i t o n the wall progressively o b s t r u c t s transmission of the r a d i a t i o n f r o m the plasma. H e n c e , a f t e r 100 to 200 d e t e r m i n a t i o n s , d e p e n d e n t o n t h e thickness of the deposit, t h e t u b e is r e m o v e d and cleaned b y soaking i n 1:l hydrochloric acid overnight, followed b y rinsing w i t h distilled water. Optimization of Microwave Power. A n increase i n m i crowave power f r o m 30 t o 50 W l e n g t h e n s the p l a s m a f r o m 6 t o 9 c m a n d increases t h e i n t e n s i t y of h e l i u m lines all along i t s length, but decreases the emission i n t e n s i t y of Zn at 2138.56 i(.T h e m e a s u r e m e n t of the emission signal of a c o n s t a n t a m o u n t of zinc at t h i s wavelength along the plasm a for t h r e e d i f f e r e n t power levels reflects t h e i n t e n s i t y d i s t r i b u t i o n along the p l a s m a ( F i g u r e 3). S i n c e zinc e m i s sion is localized a l m o s t at t h e leading edge of t h e p l a s m a , i.e., t o w a r d the s a m p l e source, only the u p p e r half of the p l a s m a w a s examined. Localization of emission m a x i m a to the leading edge of t h e discharge has been r e p o r t e d previously for an a r g o n p l a s m a at a t m o s p h e r i c p r e s s u r e ( 3 ) , and has b e e n a t t r i b uted to plating o u t of m e t a l s on t h e wall of t h e discharge t u b e so that t h e y a r e n o t carried f u r t h e r i n t o t h e p l a s m a . In s u b s e q u e n t investigations, all m e a s u r e m e n t s were performed at a power of 30 W, sufficient to m a i n t a i n a stab l e discharge and p e r m i t t i n g o p e r a t i o n w i t h o u t cooling either the cavity or t h e discharge t u b e . Optimization of Helium Flow Rate. The i n t e n s i t y of t h e Zn 2138.56-A line w a s m e a s u r e d at various h e l i u m flow rates. M a x i m u m i n t e n s i t y was o b t a i n e d at a flow r a t e of 0.48 l./min, m e a s u r e d at a t m o s p h e r i c pressure, and t h i s flow r a t e was used t h r o u g h o u t t h e s e experiments. U n d e r t h e s e conditions, t h e p r e s s u r e i n the discharge tube w a s 3.8 Torr ( m e a s u r e d b y a M c L e o d gauge). S i n c e the p r e s s u r e i n the discharge t u b e increases as a f u n c t i o n of increased heliu m flow r a t e , t h e s e t w o p a r a m e t e r s could n o t be varied ind e p e n d e n t l y a n d , hence, t h e i r individual effects on the emission intensity of d i f f e r e n t m e t a l s could n o t be assessed.
g 4 UJ
z w
$2
0
0 20
IO
0
30
4
12
8
16
[KCI]. mM
DISTANCE FROM CAVITY CENTER, rnrn
Figure 4. Effect of alkali salts on the intensity of Zn 2138.56
A
Intensity, arbitrary units. Zinc concentration, 0.02 pg/ml. Added salts: ( 0 ) none; (m) IOmMKCI; (A)10mMLiCI; (0)IOmMNaCI; (A)4mMCsCl
Figure 5. Effect of variation of the concentration of potassium chloride on the intensity of Zn 2138.56-A at 22.5 mm from the cavity center Intensity, arbitrary units. Zinc concentration, 0.02 @g/ml
~
Table I . Distance from Cavity Center of Maximum Emission Intensity for Various Elements in the Presence and Absence of Potassium Chloride
Table 11. Enhancement Effect of Various Salts on the Intensity of Zinc and Manganese Lines
Distance from cavity center, m m Ionization
Element
Ca Pb
Mn Mg cu co Fe Cd Zn
Uavelength,
x
4226.728 4057.820 4030.755 2852.129 3247.540 3453.505 3719.935 2288.018 2138.56
potential,
Without
V
addition
KC 1
6.11 7.42 7.43 7.64 7.72 7.86
27.5 30.0 30.0
30 .O
7.87 8.99 9.39
30.0 30.0 27.5 25.0 25.0 27.5
17.5 29 .O 27.5 26.0 27.5 27.5 17.5 17.5
Optimization of the Power Level during Sample Vaporization. The peak intensity of Zn 2138.56 increases with an increase in the charging voltage of the condenser, Le., with an increase in power supplied to the filament. However, when the charging voltage exceeds 8.3 V, the tantalum filament itself begins to vaporize and this metal is deposited on the wall of the discharge tube. As a consequence, successive analyses a t this voltage lead t o a progressive diminution in transmission of emitted radiation. Based on these experimental results, the charging voltage in the present study was fixed a t 7.5 V for all elements determined. The resultant temperature of the filament could not be determined accurately, since the changes occur too rapidly. The maximum temperature, however, was found to be between 1800 and 2000 “C, as measured by means of a Leeds and Northrup optical pyrometer. Matrix Effects. Since biological matter can contain unusually high concentrations of sodium and potassium, the effects of these ions were examined in detail. The chlorides of all of the alkali metals not only enhance the intensity of Zn 2138.56-A substantially (Figure 4) but also affect the distribution of intensity along the plasma (Figure 4). Indeed, with potassium and sodium chlorides, Zn emission is no longer confined to the leading edge of the plasma, and the position of maximal intensity is moved down the tube much closer to the cavity center (Figure 4). Examination of the effect of potassium chloride on a variety of elements reveals that, in some instances, a similar effect is observed, Le., the addition of potassium chloride shifts the position of the emission maximum toward the cavity, while in other cases no effect is observed (Table I). The positions of maximal intensity, of course, are different for the different elements and, while there may well be a simple relationship between the position of the maximum and the ionization
Added salts, 0,008.M
CaCl KC1 NaCl BaC1, LiCl CaC1, NHdCl
Enhancement factor
Ionization potential, V
Zn 2138.56-i.
3.89 4.34 5.14 5.21 5.39 6.11
3 6 5 0.6 6 0.8
...
1
Mn 4030.755-.i
5
1000 1000
160 1600 200 18
potential of the metal ( 1 3 ) ,the data obtained here (Table I) would have to be extended to examine this proposition more definitively. As the concentration of potassium chloride is varied, the intensity of Zn 2138.56-A rises rapidly between 0 and 2 m M of potassium chloride, is constant between 2 and lOmM, and gradually decreases above these concentrations (Figure 5 ) . Table I1 summarizes the maximal effects of various salts on the intensity of Zn 2138.56- and Mn 4030,755-A. The enhancement factor denotes the ratio of intensity in the presence and absence of salt a t that position of the discharge tube which yields maximal intensity for each element. The addition of the alkali metals markedly increases the intensity of the manganese line, though a simple correlation between the degree of enhancement and the ionization potential of the matrix element is not apparent. Figures 6 and 7 are oscillograms obtained for solutions of zinc and copper in the absence, above, and presence, below, of potassium chloride. In each oscillogram, the lower, slightly curved trace shows the time course of the voltage applied to the filament and illustrates the delay time of the signal, if the initiation of heating is chosen as the reference. The enhancement of the signal by potassium chloride is clearly apparent. Moreover, for copper, the addition of potassium chloride reduces the delay in attainment of maximal intensity from 75 to 30 msec. Analogous decreases in delay time are observed for magnesium and manganese but those for zinc and cadmium are insignificant. Interestingly, the enhancement factors for copper, magnesium and manganese are significantly higher than for zinc and cadmium. Apparently, potassium chloride may serve as a “carrier” in vaporization of relatively nonvolatile metals. T h e presence of potassium chloride allows the use of a wider power range to achieve desolvation and results in a better signal-to-noise ratio for some of the elements. Figure 8 exemplifies the effect of variation of the desolvation current on the intensity of Fe 3719.935-w for a desolvation time of 1 min. A t desolvation currents less than 1.8 A, solANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975 * 1031
m
I2l L0
intensity, arbitrary units. Sample solutions are: (B) 0.3 fig/ml Fe; ( 0 )0.03 fig/ml Fe and 8mM KCI
Table 111. Detection Limits for Various Elements in the Presence of Potassium Chloride Detection limits
Element L\‘a\,elength, A
Cd
Zn Pb
Fe
co Mg Mn Figure 6. Oscillograms of signal of Zn 2138.56-A for solutionsi containing ( a )0.01 pg/ml Zn and ( b ) 0.01 pglml Zn and 4mMKCI Lower traces show the voltage on the filament. Amplifier gain, 0.5 X 10-5. Signal, 100 mV/div. Filament voltage, 5 V/div. Time base, 20 mseddiv
Flgure 7. Oscillograms of signal of Cu 3247.540-A for solutions containing (a) 1.0 /rg/ml Cu and (6)0.01 pg/ml Cu and 8mMKCI I
Amplifier gain, ( a ) 1 X lo-’ and ( b ) 0.5 X as those in Figure 6
1032
Other conditions are same
ANALYTICAL CHEMISTRY, VOL. 47,
NO. 7, JUNE 1975
cu
2288.018 2138.56 4057.820 3719.935 3453.505 2852.129 4030.755 3247.540
ud m l
ng
0.0005 0.0003 0.01 0.002 0.007 0.00005 0.0003 0.0002
0.003 0.002 0.05 0.01 0.04 0.0003 0.002 0.001
Concn Eh h a n c e range of ment KC1,m \t factor
1-5 2- 10 7- 12 6- 12 6- 12 6- 12 7- 12 4- 12
3 6 30 50 200 500 1000 1000
vent still remains on the filament after this period. In the absence of potassium chloride, the intensity of the iron line suddenly decreases when the desolvation current is raised above 2.2 A, while in the presence of 8mM potassium chloride, a constant signal is obtained between 1.8to 3.2 A. Addition of known amounts of potassium chloride to the sample would be expected to suppress potential interference of other constituents of the matrix and, indeed, in the presence of 4mM potassium chloride, sodium and calcium chloride up to 3mM and hydrochloric acid up to 0.3M do not affect the intensity of zinc emission. Based on these considerations and results, the addition of between 4 and lOmM potassium chloride to the sample was adopted as a standard part of the analytical method. Limits of Detection. Potassium chloride enhances the intensity of all elements examined and, hence, affects the detection limits obtainable (Table 111). The detection limit is defined as that concentration or absolute amount required to result in a signal three times greater than that of the standard deviation of the background noise level. All measurements were performed using exactly the same operating conditions but optimizing the concentration of potassium chloride. No effort was made to seek ideal conditions for each element beyond this by varying yet other parameters. Hence, the possibility exists that yet further refinement is possible in some cases, since only zinc, which served as the “standard of reference”, was investigated intensively. The concentration range of potassium chloride over which the spectral intensity is constant and the corresponding enhancement factor are also shown in Table 111. Precision was measured with 5-pl samples containing 0.01 pg/ml of zinc in 4mM potassium chloride. For 17 replicate determinations, the relative standard deviation of both the peak and integral intensities is 4.4%, about 1%being attributable to pipetting error. For all of the elements listed, calibration curves were prepared to span a range of 3 to 4 decades of concentration
Table IV. Microwave-Induced Emission a n d Atomic Absorption Spectrometric Determination of Zinc Stoichiometry i n Zinc Metalloenzymes 9-atom Znlmole of protein
-~ ~
Microwave Zinc metalloenzymes
Bovine carboxypeptidase A Human carbonic anhydrase Horse-liver alcohol dehydrogenase E , coli alkaline phosphatase a
Atomic
emissiona abrorptionb
1.0
1.1 4.2 3.7
1.0 1.1 3.9 3.6
-0.lpgofenzyme.b -100pgofenzyme.
using standard solutions containing 4 to lOmM potassium chloride. At lower concentrations, the log-log calibration curves for copper, manganese, magnesium, and zinc exhibit an upward curvature, probably due to contamination of both potassium chloride and water with trace elements, estimated to be of the order of 0.0001-0.001 wg/ml, as deduced from curve fitting to yield linear calibration curves. In the absence of matrix, the calibration curves of iron and cobalt exhibit very steep slopes, which are converted to unit slopes on addition of potassium chloride. Similar effects have been reported in an argon plasma under atmospheric pressure (3, 16), though the underlying mechanism is not understood. The enhancement effect of alkali metals in microwave discharge has been described (6, 16) and the underlying mechanisms have been discussed (16). The lack of correlation between the ionization potential of a given metal and the capacity of potassium chloride to enhance its emission indicates that the mechanisms involved must be more complicated than has been assumed (6, 16, 17). The nature of the vaporization process and pertinent chemical reactions such as oxidation should be considered in delineating a possible basis. Such studies are now in progress. Analytical Applications. The microwave-induced emission spectrometric method is exceptionally well suited for the analysis of biological materials. Problems which require extreme sensitivity of detection to determine very low concentrations of metals in minute amounts of material have generally been inaccessible by conventional methods. Since microwave-induced emission spectrometry can detect pg levels of metals, this procedure may be expected to give new direction within future biological research efforts. However, when measuring metals in biological samples, the organic and inorganic constituents of the samples along with buffers, inorganic and organic reagents employed must be considered as constituting potential sources of interference. When specific macromolecules such as metalloenzymes are analyzed, high concentrations of these agents must be removed before accurate metal analyses can be performed. We have therefore established the accuracy of the microwave-induced emission spectrometric method on biological material by analyzing highly purified metalloenzymes of known zinc stoichiometry readily available to us. In particular, we have measured four zinc enzymes, Le., alcohol dehydrogenase of horse liver, alkaline phosphatase of E. coli, carboxypeptidase of bovine pancreas, and carbonic anhydrase of human erythrocytes. These enzymes were either dialyzed vs. metal-free buffers containing 10mM KCl and lOmM Tris, or their crystals were dissolved in 1mM HC1 containing lOmM KC1. Table IV compares the results of zinc analyses of each as determined by microwave excitation with those obtained using atomic absorption spectrometry, which itself had been shown previously to be in excellent agreement with data obtained by emis-
sion spectrographic and dithizone extraction methods (18, 19). For atomic absorption analyses of these enzymes, minimally 1OO-wg samples were required, while the microwave excitation procedure required only 0.1 to 2 pg. Yet, even though 1000 times less protein is needed for microwave excitation analysis, excellent agreement was obtained. Many metalloenzymes cannot be isolated readily in large quantities as these can and, hence, analysis by conventional procedures would not be possible. The analysis of the reverse transcriptase from avian myeloblastosis virus is an excellent example of the power of the method with respect to such problems (18, 19). It is available only in miniscule amounts, and must be stored a t liquid NP temperatures in 20% glycerol, 0.3M KC1, 0.01% Triton X-100, 0.05M imidazole, and 1mM dithiothreotol to remain stable and viable. In our studies of this enzyme, these metal emission quenching agents were removed by gel exclusion chromatography on G-100 prior to microwave excitation analysis. The enzyme was eluted with a buffer containing 0.01M KC1,lmM dithiothreotol and 0.001% Triton X-100. At these concentrations, none of these agents interfere with zinc, iron, copper, or manganese determinations. Moreover, the enzyme is stable in this buffer for one hour at 4 ' . While thus removing agents which interfere with microwave excitation metal analysis, the micro gel exclusion chromatography employed simultaneously served to purify the enzyme, removing any low molecular weight protein contaminants. Analyses were performed on 5-pl samples containing from 1-1.5 wg of protein. In this manner, this reverse transcriptase has been shown to contain 1.8 g-atoms of zinc per molecular weight of 180,000, while copper, iron, and manganese are absent (18, 19). More recently, the reverse transcriptases from mammalian tumor viruses, e.g., those from the murine and simian leukemic viruses have been similarly shown to contain stoichiometric amounts of zinc (20). Forty years ago Policard (21) first attempted to perform microemission spectrometry on tissues and single cells which proved beyond the then available technical facilities. The methods, however, were refined and extended subsequently for the analysis of inclusions in steel and mineral splinters in rocks (22). In the biological field, however, repeated attempts with steadily improved equipment have remained futile, largely owing to the combination of the low metal concentrations in the minute amounts of biological matter found in individual cells when combined with available methods with relatively high detection limits demanding large amounts of material for analysis. Most recently, laser excitation spectroscopy has shown promise for this purpose though the attending problems have not as yet been fully overcome (23). Our preliminary data show that a micro-analytical system based on microwave excitation spectrometry has all the features required for the determination of the metal content of single cells, single crystals of metalloenzymes, and other metal-containing materials. Its utilization in the study of biological micro- and ultra-structure is currently under investigation. ACKNOWLEDGMENT The cooperation of Monsanto Research Corporation in providing instrumentation is gratefully acknowledged. LITERATURE C I T E D (1) A. J. McCormack, S. C. Tong, and W. D. Cooke, Anal. Chem., 37, 1470 ( 1965). (2) C. A. Bache and D. J. Lisk. Anal. Chem.. 37, 1477 (1965). (3) J. H. Runnels and J. H. Gibson, Anal. Chem., 39, 1398 (1967). (4) K. M. Aldous. R. M. Dagnall. 8. L. Sharp, and T. S. West, Anal. Chim. Acta, 54, 233 (1971).
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(5) K . Faiigatter, V. Svobcda, and J. D. Winefordner, Appl. Spectrosc., 25, 347 (1971). (6) H. Kawaguchi, M. Hasegawa, and A. Mizuike, Spectrochim. Acta, Part 8,27, 205 (1972). (7) H. Kawaguchi, M. Hasegawa. and A. Mizuike, J. Jpn Spectrosc. Soc., 21, 36 (1972). (8) H. Kawaguchi, T. Sakamoto, and A. Mizuike. Ta4nta. 20, 321 (1973). (9) F. E. Lichte and R. K . Skogerboe, Anal. Chem., 45, 399 (1973). (10) C. A. Bache and D. J. Lisk, Anal. Chem., 30, 786 (1967). (11) D. N. Hingie, G. F. Kirkbright, and R. M. Bailey, Talanta, 16, 1223 (1969). (12) H. E. Taylor, J. H. Gibson, and R. K. Skogerboe, Anal. Chem., 42, 1569 (1970). (13) G. W. Wooten, E. L. Brown. and J. F. Moon, private communication (1973). (14) K . W. Busch and T. J. Vickers, Spectrochim. Acta, Part 8, 28, 85 (1973). (15) D. E. Nixon. V. A. Fassei, and R. N. Kniseiy, Anal. Chem.. 46, 210 (1974).
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RECEIVEDfor review November 18, 1974. Accepted February 7 , 1975. This work was supported by Grant-in-Aid LH-94 from the International Lead Zinc Research Organization, Inc.
Simultaneous Determination of Seven Trace Metals in Potable Water Using a Vidicon Atomic Absorption Spectrometer Kenneth M. Aldous, Douglas G. Mitchell, and Kenneth W. Jackson Division of laboratories and Research, New York State Department of Health, New Scotland Avenue, Albany, NY 12201
A multichannel atomic absorption spectrometer Is used for the determination of Zn, Cd, NI, Co, Fe, Mn, and Cu in potable waters. Metals are chelated with ammonium pyrroiidine dithiocarbamate at pH 4, and a 10-fold concentration is achieved by extracting into methyl isobutyl ketone. The organic phase is aspirated into an air-acetylene flame, and atomic absorption is measured simultaneously at the resonance lines of these elements by disperslng a 168-nm region of the lamp and flame spectrum across a vidicon array detector. Detection limits from 0.004 to 0.02 pg/mi have been obtained, with dynamic ranges up to 100 and relative standard deviations of 3 % at optimum concentrations. This performance, though poorer than by conventional singlechannel atomic absorption spectrometry, is adequate for routine monitoring of public water supplies and most waste waters.
Potable water contains metals a t both moderate and trace concentration levels. The major constituents, Ca, Mg, Na, and K, are present a t from 1 to 250 Kg/ml; other metals are below 1 kg/ml. The former group can be directly determined by conventional flame techniques using direct nebulization without pretreatment, while the latter group requires preconcentration. Atomic absorption (AA) spectrometry is the preferred analytical technique for most metals, with trace metals preconcentrated by ion exchange ( I ) , partial evaporation (Z), or chelation and solvent extraction (3, 4 ) . Atomic absorption methods are rapid, simple, precise, and accurate, and interferences seldom occur. However, with a conventional single-channel spectrometer, elements must be analyzed in sequence, an increasingly inefficient procedure as the number of metals per sample increases. Arc or spark source emission spectrometry is capable of simultaneously determining large numbers of elements per sample, but this technique requires a substantial capital investment and skilled spectroscopists and is not suitable for moderately sized laboratories. Several multielement spectrometers using AA, atomic emission (AE), and atomic fluorescence 1034
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spectrometry have been described in the literature ( 5 ) ,but these are not readily available, and their construction generally requires engineering skills not found in the average analytical laboratory. Vidicon television camera tubes enable the simultaneous observation of all wavelengths over a wide spectral range, and these detectors have recently been shown to be suited for both flame AA (6-8) and AE (9-11) spectrometry. Basically, part of the lamp and flame spectrum exiting from a monochromator is dispersed across a light-sensitive target to produce a charge pattern. This charge is read by a scanning electron beam, and the charge density a t each point on the target is obtained. The charge density is a function of radiative intensity, and the position is a function of wavelength. We have developed a multichannel AA spectrometer with a silicon-target vidicon detector responding in the ultraviolet region (7) and have used this instrument for the simultaneous determination of trace wear metals in used lubricating oils (8).Here we describe its further application to the simultaneous determination of seven trace metals in potable water samples. With this system, the major metals (Ca, Mg, Na, K) are not readily determined simultaneously because their principal resonance lines are spread between 285.2 nm (Mg) and 766.5 nm (K). All radiation over a wavelength range of 481.3 nm would have to be dispersed across the detector, and this would require a low-dispersion grating with impractically low resolution (7). However, the most important trace components, including Zn, Cd, Ni, Co, Fe, Mn, and Cu, have principal resonance lines between 213.9 nm (Zn) and 324.7 nm (Cu), and this 110.8-nm range can be readily dispersed onto a vidicon detector using a moderate-resolution monochromator.
EXPERIMENTAL Apparatus. The multichannel AA spectrometer, consisting of two multielement hollow cathode lamps (Jarrell-Ash, Waltham, MA), air-acetylene burner/nebulizer, 0.25-meter Ebert monochromator, and silicon-target vidicon detector, has been described previously (7). The vidicon tube with associated electronic console was the SSRI Model 1205 Optical Multichannel Analyzer (Prince-
