350
Anal. Chem. 1081, 53, 350-354
ACKNOWLEDGMENT
(13) Mill, A. J. 6. W.D. Thesis, Imperlal Conege Science 8 Technology, London, 1976. (14) “Trace Metal Decontamination Procedures for Sample Containers”, Sheet M27; Environmental Science Assocletes, Inc.: Bwilngton, MA. (15) Subramanlan, K. S.; Chakrabarti, C. L.; Suekas, J. E.; Maims, I. S. Anal. Chem. 1078. 50, 444. 116) Meranaer. J. C.: Subramanlan. K. S.: Chaiifoux. C. Envkon. Scl. Tech&/. ‘1070, 73,707. (17) Shendrkar, A. D.; Dharmarajan, V.; Walker-Merrlck, H.; West, P. W. Anal. Chlm. Acta 1078, 64, 409. (18) Fukai, R.; Mwray, C. N.; Huynh-Ngoe, L. Estuarine Coastal Mar. Scl. 1075, 3, 177. (19) Florence, T. M. WaterRes. 1077, 7 7 , 681. (20) Pddoskl, J. E.; Glass, G. E. Anal. Chlm. Acta 1078, 707, 79. (21) Martin, J.4.; Meybeck, M. Mar. Chem. 1070, 7, 173. (22) WHson, A. L. “Concentrations of Trace Metals in River Waters: A Revlew“; Technical Report TR16; Water Research Center: Medmenham, Bucks., England, 1976. (23) Janssens, M.; Dams, R. Anal. Chlm. Acta 1073, 65, 41. (24) Laxen, D. P. H.; Harrison. R. M.. unpublished results, 1979.
The authors are grateful to J. M. Walsh for her assistance with the atomic absorption analysis.
LITERATURE CITED Batley, G. E.; Gardner, D. Water Res. 1077, 7 7, 745. Patterson, C. C.: Sew, D. M. NBS. Spec. Publ. ( U . S ) 1076, 422, 321. Karin, R. W.; Buono, J. A.; Fasching. J. L. Anal. Chem. 1075, 47, 2296. Moody, J. R.; Lindstrom, R. M. Anal. Chem. 1077, 49, 2264. Batley, G. E.; Gardner, D. Estuarine Coastal Mar. Scl. 1078, 7 , 5% Marchant, J. W.; Klopper, 6. C. J. Oeochem. Explor. 1078, 9 , 103. WhReside. P., Ed. “Atomic Absorption with ElectrothermalAtomizatkm; b e Unicam Ltd.: Cambridge. England, 1977; p 14. Baler, R. W. J. Envlron. Qual. 1077# 6 , 205. Nurnberg, H. W.; Vaienta, P.; Mart, L.; Raspar, 6.; Sipos, L. 2. Anal. Chem. 1078, 282, 357. Sugel, S. F.; Healy, M. L. Mar. Chem. 1078, 6 , 291. Department of the Envkonment “Lead in Potable Waters by Atomlc p 6. Her Ma/esty’s Office: don, 1976; Spectrophotomew”; Klng, W. G.; Rodrigues, J. M.; Wai, C. M. Anal. Chem. 1074, 46, 771.
. .
RECEIVED for review February 15, 1980. Accepted October 15,1980. This work was supported by a research grant from the United Kingdom Science Research Council.
Photometric Instrument with Automatic Switching between Photon Counting and Analog Modes Vance J. Nau and Timothy A. Nieman’ School of Chemical Sciences, University of Illinois, Urbana, Illinois 6 180 1
The signal output from a photomultiplier tube (PMT) can be processed by either photon counting (PC) or direct current amplification (DC), where DC also includes the lock-in amplifier and noise-voltage (high pass filtering) techniques. Each method has distinct regions of superiority (PC at low levels and DC at high light levels), thus making each an ideal complement of the other. PC techniques have, for several years, been recognized as superior to DC techniques at low light levels (due to fundamental signal-to-noise ratio considerations) and additionally at intermediate light levels where the direct digital nature and freedom from drift of PC provide practical advantages (1-9). Recently, several papers have focused on improved systems for photon counten (10, 11),characteristics of photon counting systems (12),and optimal strategies for photon counting (13, 14). The principal disadvantage of PC is the loss of linearity due to pulse pileup a t high photon arrival rates (typically lo6-lo6 Hz). For an RCA 1P28, this rate corresponds to 10-8-10-7 A (1,15),which is a severe limitation since the PMT response function is linear to 5 x 10” A (15). Several methods have been proposed to extend the linear range of a particular PC system, including dead time compensation (16) and count-loss correction (17).These approaches can be of use in certain cases but are capable of only limited extension of the linear range. DC techniques are therefore superior to PC techniques a t high light levels where PC becomes nonlinear and in situations where high precision (RSD < 0.1%) is desired (9). It is apparent that no single photometric technique (neither PC nor DC) is adequate to provide optimum performance over the wide range of outputs possible from a PMT. A number of manufacturers have already recognized the complementary nature of PC and DC techniques by combining independent PC and DC photometers into a single housing. (These are Model DPC-2 digital photometer, SPEX Industries, Metuchen, NJ; Model 1140 quantum photometer, Princeton Applied Research, Princeton, NJ; and Model 126 photometer, 0003-2700/81/0353-0350$01.00/0
Pacific Precision Instruments, Concord, CA.) Since these instruments do not yet offer normalized data output or provide automatic mode selection, the user must manually select the desired technique prior to initiation of a measurement. The need to manually change modes of operation negates the versatility and extended linearity that would otherwise be possible. We have developed a combined PC/DC photometer which takes full advantage of the sensitivity and stability inherent in PC and of the extended linearity and high precision available at high photon flux allowed only by a DC technique. The PC and DC portions are interconnected by hardware logic, which allows real time ( (PC), and (DC), >> (DCId, conditions which are easily satisfied), the CF reduces to C F = (pC)t/(DC)t
(2)
If (DC), can be set equal to an integral power of ten (i.e., 10, 100,1000, etc.), then CF = (PC),, ignoring any decimal place. The (PC), value is then used to preset a series of BCD down counters to adjust the DC time base proportionally to correct for the unequal sensitivity of the DC system. In the example provided by Table I, the lower sensitivity of the DC system (DC count rate of 1230lunit time compared to a PC count rate of 6494lunit time) is compensated by extending the DC time base by a factor of 5.280 (649411230 = 5280/1000 = 5.280). Up to 2 orders of magnitude can easily be normalized with this method by varying the predetermined number of DC counts allowed to occur during the calibration cycle. For example, if the sensitivity of the DC system were decreased such that only 123 counts (instead of 1230) were observed in counting period T and calibration terminated a t 100 DC counts (instead of lOOO), the CF then becomes 64941123 = 5280/100 = 52.80. Thus, the conditions selected for the calibration cycle depend upon the relative sensitivities of the PC and DC systems. The instrument also features an autoranging time base to ensure that a maximum number of counts are displayed a t all times. To avoid any undesirable signal averaging, as in kinetic rate measurements, the user can select a maximum time base beyond which the instrument cannot autorange. It should be stressed that although the instrument will operate with present hardware control, all of the control decisions may be externally imposed by either manual or computer intervention. EXPERIMENTAL SECTION PC Linearity. The linearity of the photon counter was established with the method described by Hayes et al. (17) for the determination of effective dead time (p') using (3) In V/O = In (Al/k) - ( A d O / k where f is the observed PC flux, AI is the discriminator efficiency for a single photon event, and k is the proportionality constant between the true PC flux, F, and the PMT current, I ( I = K F ) . The regression of In V/n on I allows the determination of p' and the uncertainty of that estimate from the slope and intercept and their respective uncertainties ( 1 7 ) .
The calibration factor, CF (where CF represents a simultaneous measurement of the ratio f/O, was measured as a function of light intensity. Each point consisted of the average of 200 separate determinationsof CF and a 20-5 average of the PMT current. An LSI-11 computer system was used to monitor the operation of the instrument and collect and process the required data. All measurements were conducted by use of a Hamamatsu R372 PMT operated at IO00 V, and a Heath Dzlamp source. The video amplifer gain was set at 100 to maintain adequate band-pass and avoid amplifier saturation when operated at higher gains with 1004 source resistance (see Figure 2). DC Linearity. Linearity of the DC system was established by comparing the VFC output frequency to the known input current (supplied by an ordinary carbon dry cell and a large variable resistor). The current was monitored continuously and found to remain constant over the time period of the experiment. Calibration Factor. The CF was measured on an hourly basis for a period of 5 h to determine long-term stability. These values consisted of the average of 100 consecutive readings (one every 50 ms collected under computer control. Precision for single measurements and for a series of measurements is compared. INSTRUMENT PERFORMANCE DC Linearity. No nonlinearity could be observed over the anticipated operating range of the DC system (100 nA-100 PA). A graph of output DC count rate vs. input current over this range yielded a correlation coefficient of 0.999 99 both for a linear and for a log-log plot. The upper linear range of the DC circuit depends upon the feedback resistor (Rf) of the operational amplifier (Figure 2). With an Rf of 100 kR the OA sensitivity function is 100 pV/nA for an overall sensitivity (OA-VFC) of 10 counts s-l nA-', which extends the linearity of the PC/DC photometer to the maximum output signal (100 PA) for the P M T used throughout these experiments (Hamamatsu R372). Rf is switch selectable to allow DC operation in other current ranges. Effects of P C Discrimination Setting. The original goal was not to build the fastest PC circuit but to integrate PC and DC circuitry into a single system which allows easy and complete utilization of the combined advantages of PC and DC techniques. Even though thee primary purpose of this article is not to report on the development of another PC system, the performance of the T T L comparator should be examined. Darland et al. (12) have shown the effect of the discriminator threshold level setting ( v d ) on the observed count rate, f, at various PMT voltages for several different PMTs, including the RCA 1P28. We have observed very similar results for the Hamamatsu R372 P M T (which is the equivalant of the RCA 1P28A),with the exception that the R372 has a lower dark count rate than the 1P28. With our system, the observed count rate (either signal or dark counts) shows a sharp increase at Vd settings less than approximately 40 mV. This sharp increase results from counting secondary emission noise pulses originating at one of the dynodes and not at the photocathode. With no PMT input to the PC circuit, false counts (resulting from noise generated by the PC circuit) are not observed a t Vd levels greater than 14 mV. Measurement of the signal to noise ratio as a function of the discriminator voltage yielded results similar to those obtained by Niemczyk (13). The expected dependence of SNR on N1/2 (where N is the observed number of PC counts) was observed at all Vd settings above approximately 40 mV. The rapid decline in the observed SNR a t lower Vd settings can be attributed to the noise from secondary emissions. Therefore, 40 mV is indicated as the optimum Vd setting for both sensitivity and SNR. P C Linearity. Since the CF is the simultaneous ratio of f/Z,any nonlinearities in f will be reflected in CF. Accordingly, CF can be used as a very accurate means of establishing the PC response as a function of I; such a plot is shown in Figure
ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981 353 OBSERVED PC COUNT RATE (Hz)
Table 11. Calibration Factor Stability hour resulta i: u hour 0 1
2 3
52.66 52.5052.40 52.45
i:
0.0108
f 0.0387 i: i
0.0168 0.0361
4 5
RSD
resulta
f
u
* *
52.48 0.0374 52.53 0.0180 52.50 f 0.0887 0.17%
a In each case the calibration cycle lasted for 100 DC counts.
I l l
16’
I
I l l 1
I
1
10-6
1
1
1
I
IO+
PMT CURRENT ( A I
Flgure 5. Effect of pulse pileup on the measured CF. CF values are not adjusted for the +4 prescaler; PC count rates have been corrected by this factor to reflect the measured anode pulse rate. The arrow indicates the PC count rate at which the photometer would change modes (if enabled). R, = 1 MQ for these measurements.
5. (The CF must be corrected for the prescaling factor of 4 to obtain an accurate estimate of the PC linearity.) The observed PC to DC sensitivity ratio (CF) is constant at about 5.2 a t low count rates but rapidly decreases as P C dead time losses become significant. The effective dead time, p’, was calculated according to the method of Hayes et al. (17)to be 31.18 f 0.04 ns, which is comparable to values recently reported for PC systems ( 1 0 , I I ) . The least-squares fit of the line defined by the regression of In V / l , on I (see eq 3) gave a correlation coefficient of -0.9999 and a standard error of estimate of 0.00039. The fit clearly indicates that the PC circuit behaved as expected with no observed anomalies due to the use of T T L logic. Calibration Factor. The precision of CF for any single determination is better than simply WI2,where N is the number of PC counts recorded during the calibration cycle. Since CF represents the simultaneous ratio of f / I (comparable to a dual-beam-in-space spectrophotometer) the actual precision is determined by the degree to which the DC circuit can simultaneously track the PC circuit (Le., respond to the same changes in instantaneous photon flux). The ideal case would be two PC circuits simultaneously counting “all the pulses’’ from a single PMT; there could be no deviation if each were, in fact, counting all the pulses. However, the DC circuit is limited by the i-to-V and the VFC circuits’ response times. For a DC sensitivity of 10 Hz/nA (Rf = 100 kQ),the observed standard deviation for any single determination of CF is about f0.6N’12-a significant improvement over that predicted by considering only photon statistics. Considering a CF of 52.80 (5280 PC counts in the same time as 100 DC counts-a ratio of 52.80) which is obtained from a 1 4 prescaler for a total of 21120 PC counts per calibration cycle, the typical precision for one measurement (no manual or computer averaging) would be f 2 2 counts out of 5280 or 0.4% RSD. Increasing the gain of the DC circuit (R, = 1 MQ) increases the response of the VFC and improves the precision of the CF to f0.5W/2 but also reduces the upper linear range of the overall PC/DC system. The usual operating conditions (Rf = 100 kQ) allow utilization of the full operating range of the PMT. The long-term stability of the CF is very good, as is seen in Table 11. Each measurement represents five repetitions of the average of 100 determinations of CF which were collected under computer control. The average and the standard deviation are indicated for each set of measurements, as well as the overall average and the standard deviation for that
average. No adjustments were made to any component (PMT voltage, v d , or DC offset) over the 5-h test period. Even though the precision and stability are excellent (f0.17% RSD over a 5 h period), the cyclic appearance of the drift implicates the DC circuit stability ( l / f noise) as the limiting factor; the fact that the uncertainty in the average (f0.0887) is much greater than the pooled uncertainty for the individual measurements (f0.0314) also indicates the presence of long-term drift. Similar precision can be obtained on a day-to-day basis if v d , the P M T voltage, and the DC offset are monitored and adjusted as necessary, but it would be much easier simply to recalibrate the instrument on a daily basis, or whenever circuit drift is detected. Calibration is conveniently accomplished in less than a second. PC/DC Operation. For both PC and DC measurements, the precision shows the expected MI2dependence provided that the two measurements extended over the same period of time. Using the CF to extend the DC time base results in the DC measurement having a standard deviation lower than the equivalent PC measurement by a factor of (CF)1/2. For example, with a CF of 5.280 the DC measurement would take 5.28 times longer than the PC measurement and the DC precision would be improved by a factor of 2.3. However, since the user always knows the mode of operation and the value of CF, the precisions can easily be compared, if so desired. Additionally, it is possible to impose a CF value of unity so that PC and DC measurements last for the same period of time. The dead time of 31.18 ns gives a calculated nonlinearity of 1.2% at a PC count rate (before prescaling) of 400 kHz. Assuming that the PC/DC photometer will be allowed to automatically change modes of operation (from PC to DC and vice versa) at that count rate and that the CF has been carefully determined (within f0.2%) in a region where both PC and DC are linear, the instrument response would be linear (less than 1.4% nonlinearity) over the entire operating range of the P M T used (to signal levels greater than the equivalent of a 120-MHz rate). The linearity could be improved to better than 1% (without sacrificing range) by substituting one of the somewhat faster PC systems recently published (10, 11) for the one currently being used. However, PMTs have finite pulse widths, typically 0.8-5 ns; therefore even if an infinitely fast PC system could be built, its performance could not match the linearity of our combined PC/DC system, since an effective dead time of less than 0.12 ns is needed to yield 1.4% nonlinearity to 120 MHz count rates. CONCLUSIONS PC techniques offer the best sensitivity, SNR, and stability a t low light levels while DC techniques are required a t high light levels and when high precision is required. We do not believe that building faster PC systems is necessarily the correct approach to solving the linearity problem of PC at high light levels, particularly if it involves deoptimization of the PC system a t low light levels. Perhaps a better approach would be to concentrate on maximizing the advantages which PC has at low light levels and to incorporate a DC system to extend the upper linear range. The instrument described successfully combines the advantages of PC and DC into a
354
Anal. Chem. 1981, 53, 354-356
single, easy-to-use unit. The overall modular design allows functions to be added as needed, such as a digital-to-analog converter card for recorder output or a peak maximum/peak area detector card. Exclusive of enclosure and power supply, the total cost was less than $500.
LITERATURE CITED Malmstadt, H. V.; Franklin, M. L.; Horlick, G. Anal. Chem. 1972, 44, 63A-76A. Franklin, M. L.; Horlick, G.; Malmstadt, H. V. Anal. Chem. 1969, 4 1 , 2-10. Ingle, J. D., Jr.; Crouch, S.R. Afl8l. Chem. 1972, 44, 777-784. Nakamura, J. K.; Schwartz, s. E. ~ p p l opt. . 1068, 7, 1073-1078. Alfano, R. R.; Ockman, N. J . Opt. Soc. Am. 1968, 58, 90-95. Jones, R.; Oliver, c. J.; Pike, E. R. App/. Opt. 1971, 70, 1673-1880. Murphey, M. K.; Ciyburn, S. A.; Veillon, C. Anal. Chem. 1973, 45, 1468- 1473. Ingle, J. D., Jr.; Crouch, S . R. Anal. Chem. 1972, 4 4 , 785-794. Hayes, J. M.; Schoeller, D. A. Anal. Chem. 1977, 49, 306-311. Dariand, E. J.; Hornshuh, J. E.; Enke, C. G.;Led, G. E. Ana/. Chem. 1979, 51, 245-250.
Borders, R. A.; Birks, J. W.; Borders, J. A. Anal. Chem. 1980, 52,1273-1278. (12)Dartand, E, J.; Leroi, G, E.; Enkg, C, G. Ana/, Chem. 1979, 57,
(11)
240-245. (13) Niemczyk, T. M.; Ettinger, D. G.; Barnhart, S. G. Anal. Chem. 1979, 57, 2001-2004. (14) . , Darland. E. J.: Lerol. G. E.: Enke, C. G. Anal. Chem. 1980. 52. 714-723. (15) RCA Photomuttiplier Manual; RCA Corp.: Harrison, NJ, 1970. (16) Ash, K. C.; Plepmeier, E. H. Anal. Chem. 1971, 43, 26-34. (17) Hayes, J. M.; Matthews, D. E.; Schoeller, D. A. Anal. Chem. 1978, 50,25-32. RECEIVED for review May 9, 1980. Resubmitted September 25,1980. Accepted October 28,1980. Presented in part at the 1979 Pittsburgh Conference on Analytic, Chemistry and Applied Spectroscopy (paper 218). This research was supported by the National Science Foundation (CHE-78-01614). V.J.N. is grateful for fellowship support funded by Phillips Petroleum.
Automated Determination of Mercury in Urine and Blood by the Magos Reagent and Cold Vapor Atomic Absorption Spectrometry Peter Coyle' and Thomas Hartley Division of Clinical Chemistry, Institute of Medical & Veterinary Science, Adelaide, 5000, South Australia, Australia
It has been our experience that the manual method for measuring mercury by cold vapor atomic absorption spectrophotometry involves a considerable number of reagents and repetitive manual steps which may lead to operator error, particularly when large batches have to be analyzed. The Magos reagent (1-3), used in measuring mercury in biological samples, has a significant advantage over other manual procedures ( 4 ) in that it permits the analyst to discriminate between mercury in organic or inorganic forms. One automated procedure (5) uses continuous flow digestion but is restricted to the measurement of total mercury. Another automated method (6) can distinguish between total and inorganic mercury, although only in samples that contain little organic material, e.g., water and waste water. Biological samples such as blood which contain large amounts of protein have not so far been assayed with automated methods. We describe a fast and simple pretreatment step which allows the measurement of mercury in both the inorganic and organic forms in blood by an automated technique. This method is more convenient to use than the manual method because it requires less operator skill. I t is sufficiently sensitive and accurate for screening subjects suspected of exposure to inorganic or organic mercury. EXPERIMENTAL SECTION Apparatus. The atomic absorption unit used was a Model 403 Perkin-Elmer double beam spectrophotometer fitted with a Perkin-Elmer Model 56 recorder. A mercury hollow cathode lamp was used as the light source. The gas flow cell was a Corning Eel design 7 mm i.d. and 100 mm long with quartz glass end windows. A Technicon Auto Analyser I1 proportioning pump was used to pump sample and reagents through the manifold illustrated in Figure 1. Standard Technicon flow rated pump and transmission tubings were used throughout. A Hook and Tucker A40 Autosampler I1 presented samples and wash solution to the manifold for 30 and 75 s, respectively. The wash solution was 0.1% L-cysteine in 2 M HCl. The vapor-liquid separator (see Figure 2) was assembled from Quickfit glassware and plastic tubing. The sample stream was injected through a right angled 0003-2700/81/0353-0354$01 .OO/O
glass tube (4 mm o.d., 2 mm id.), and the nitrogen stream was injected through a glass nozzle with an internal tip diameter of 0.8 mm. Both these pieces were mounted in a length of rigid polythene 9 mm i.d. and 50 mm long. Reagents. All chemicals were of analytical grade. The manifold reagents for total mercury measurements were made up at the concentrations detailed in Figure 1. The sodium hydroxide and L-cysteine were made up in glass distilled water, the SnC12-CdC12was prepared in 2 M hydrochloric acid, and the wash solution was a 0.1% solution of L-cysteine in 2 M hydrocholoric acid. The antifoam was prepared as a 10% solution of 1-octanol in ethanol. When the manifold was used for the selective measurement of inorganic mercury, the SnCl,-CdCl, reagent was replaced by a 10% solution of SnC1, in 2 M hydrochloric acid. A stock loo0 pmol L-l inorganic mercury standard was prepared by dissolving 0.2715 g of HgC12in 5% (v/v) sulfuric acid. A 1-g sample of L-cysteine was then added and dissolved before making up to a final volume of 1 L with the sulfuric acid. Portions of this.stock standard were then diluted with glass distilled water to give a series of working standards containing 0.25,0.50, 0.75, and 1.00 pmol L-' of mercury. A stock lo00 pmol L-' methyl mercuric chloride standard was prepared by dissolving 0.2511 g of the anhydrous solid in 100 mL of acetone. The final volume of this solution was adjusted to 1 L with glass distilled water. Working standards were prepared at the same concentrations as the inorganic mercury standards. Sample Preparation. Determination of Total Mercury in Urine. Urine samples and standard solutions were analyzed directly. One-milliliter aliquots were placed in sample cups on the autosampler turntable. The SnC12-CdC12reagent was used in the manifold. Determination of Total Mercury in Blood. The blood samples were Dretreated as follows. One milliliter of a 6% L-cysteine solutibn was added to 1 mL of the whole blood or aqueous standard and mixed. A 1-mL sample of 40% trichloroacetic acid was then added to each tube, and the tubes were shaken vigorously. The specimen was centrifuged for 5 min at 3000 rpm. The clear protein-free supernatant was placed in sample cups on the autosampler. The SnC12-CdC12reagent was used in the manifold. Determinution of Inorganic Mercury in Blood. Specimens were treated in the same manner as in the total mercury in blood 0 1981 American Chemical Society
