Table VI. Comparative Lipase Activity of Various Commercial Products
Activity, units/gram Procedure
steapsin lipase was used in all inhibitor determinations. I n all runs, a two minute preincubation of enzyme with inhibitor was sufficient for maximum sensitivity (Table 111, A).
of
Product Pancreatin N.F. Powder, Wilson Lot # 122790 Lipase,,Steapsin, Nutritional Biochemical Co. Li ase, Porcine bancreas, Galbiochem. Co. Li ase, Pancreatic, Kiilson Lot # 124142 Lipase, Pancreatic, Wilson Lot # 122005
FluoriLaeometric Wasem procedure (8) 482.5 716.0 2100
500 710 2110
3323
3220
2440
2450
Porcine pancreas and wheat germ lipase are inhibited by extremely small amounts of sarin (0.012 pg. per ml. for 50% inhibition). Although the porcine pancreas and wheat germ lipases are more sensitive to sarin than is steapsin, the enzyme activities are not linearly related to the sarin concentrations and the data are not as reproducible as in the case of steapsin. For these reasons,
Radiof reque ncy
0
ANALYTICAL CHEMISTRY
The results of the determination of samples of various enzymes and inhibitors are indicated in Tables IV and V. I n general, samples of steapsin and porcine pancreas lipase of concentrations 0.0250 to 0.250 and 0.00250 to 0.0500 mg. per ml., respectively, were analyzed using a 5 X 10-6M solution of dibutyrylfluorescein as substrate, with relative standard deviations of f1.6 and 1.570,respectively. Wheat germ lipase, 0.0125 to 0.200 mg. per ml., was determined using a 5 X 10-sM solution of diacetylfluorescein as substrate with a relative standard deviation of *1.5%. Acylase, 0.0060 to 0.100 mg. per ml. and a- and y-chymotrypsin, 0.167 to 1.30 mg. per ml. were analyzed with relative standard deviations of *1.9%, *2.5%, and d=1.5%,respectively. The organophosphorus compounds, sarin and Systox, 0.033 to 0.317 and 0.0660 to 0.330 pg. per ml., respectively, were analyzed with relative standard deviations of *1.5% and *2.0%. Finally, Triton X-100, in concentrations of 0.0700 to 0.500% by volume in the
PIasma
SIR- Quantitative emission spectrochemistry using flames for excitation has limitations placed on it by the relatively low energies available and by the complications inherent in the chemical nature of gases undergoing combustion. A plasma-source emission spectrophotometer has been developed which shows significantly increased sensitivity for many elements together with freedom from a number of the spectral difficulties introduced by combustion flames. The instrument makes use of the high excitation energy available in a radiofrequency nitrogen plasma to excite electronic transitions in atoms. An aqueous fog of the sample is produced by means of an ultrasonic atomization technique and passed into the plasma. A recent paper by Mavrodineanu and Hughes (3) describing the use of a similar plasma source in emission spectroscopy has come to our attention since this manuscript was submitted. These workers have used primarily a 412
RESULTS
overall solution, was determined with a relative standard deviation of i1.4%. A number of commercial lipase preparations from various sources were analyzed to test the reliability of the fluorimetric procedure, and these results are given in Table VI. The analysis of a number of standard lipase samples yielded results which agreed well with those results obtained by the procedure of Lazo-Wasem (8), using olive oil as a substrate. LITERATURE CITED
(1) Aldridge, W. N., Biochem. J. 57, 692
(1954). (2) Bier, M., “Methods in Enzymology,” Vol. I, p. 630, Academic Press, New York. 19.55. ~-~ ~ - - (3) Giang, P. A., Hall, S. A., ANAL. CHEM.23, 1830 (1951). (4) Greenstein, J. P., “Biochemists’ Handbook,” C. Long; ed.] p. 288, Van Kostrand, Princeton, 1961. ( 5 ) Guilbault, G. G., Kramer, D. X., Cannon, P. L., ANAL.CHEM.34, 1437 I
(lQ62) \-_--
(6) KrLmer, D. N., Guilbault, G. G. Ibid., 35, 588 (1963). (~, 7 ) Laskowski. M.. “Biochemists’ Handbook,” C. ’Long, ed., p. 304, Van Nostrand, Princeton, 1961. (8) Lazo-Wasem, E. A., J . Pharm. Sci. 50, 999 (1961). (9) . , hfvers. D. K.. et al.. Biochem. J. 61. 521 ”( 1955). ’ (10) Schonheyder, F., Volqvarte, K., Biochim. Biophys. Acta 6 , 147 (1950).
RECEIVEDfor review July 29, 1963. Accepted October 23, 1963.
Emissio n S pect rophoto meter
magneton-powered microwave discharge in hydrogen or helium and have obtained photographic records of the spectra of 75 elements introduced as solids into the source. With a 30-mc. source, ,spectra of various metals were obtained by spraying aqueous solutions into the gas stream. No detection limits of estimates of precision were given. Plasma Torch. The plasma generator was electronically identical to the 27-mc. torch described by Roddy and Green (4). To allow introduction of aqueous samples, the tank coil and tip area were modified as shown in Figures 1 and 2. The cross-hatched area is machined from Teflon. The atomized sample in the carrier gas enters the chamber through a 6.35-mm. hole in the side of the unit and passes up through a 32-mm. i.d. quartz tube placed inside the tank coil and connected to the Teflon unit. The torch power supply may be any commercial model-e.g., Precision Instruments Co. No. 7-
6003AV-capable of producing 3 kv. maximum, and supplying a constant input power of 500 watts to the plasma generator for sustained periods of time. The torch constructed in this laboratory operated more satisfactorily when an improved capacitive link to ground was furnished. This was conveniently accomplished by removing the atomizerburner support and sample holder from a cast-aluminum Beckman DU flame photometer burner housing, positioning the housing around the quartz tube just above the tank coil, and grounding it. The mirror in the housing offered a slight increase in sensitivity, and the housing gave eye protection against the ultraviolet radiation produced. For shielding and for protection against the lethal voltages involved, the plasma torch was completely encased in a Lucite box covered by grounded copper screening. I n appearance, the nitrogen plasma consisted of a brilliant pink ribbon of
\
B
t!
H Lucite Box=
Figure 2. Detail of Teflon chamber a t base of torch showing arrangement for sample to flow upward around torch tip
1.
tip:
Figure 3.
Plasrna
Sample enters a t A. Cooling water circulated through roncentric copper tube at B. Torch
molybdenum
light, about 3 mm. in diameter and 8 cm. high, surrounded by much less brilliant area. perhaps 2 cm. in diameter and 12 cm. in height. When the nitrogen flow is adjusted properly, there is no visible flick.ering. Atomizing System. d n y sort of atomizcr may, in Frinciple, be insertcd brtwecn the gas supply and the plasma generator. The atomizing system of the 13airtl-Atomic Model DI34 flamci photometer has been used, as has a medium-bore Beckman atomizer. Both these atomizers require rather high gas flow rates for satisfactory operatioil, whereas high stability in the plasma necessitates a low gas flow rate (about 2 liters per minute). With .such atomizers it is possible to introduce only a small portion of the sample s ,ray to the torch, and most of the atomized sample is wasted. An ultrasonic atomizing system has the dual advantages oi' high sensitivity and high efficiency. Since it does not depend on a ral)idly moving gas stream to form the droplets, the necessary low flow rates may be used. The rate of sample consumption i,; loiv (less than 0.1 ml. per minute), and a large percentage of the atomizttd sample enters the torch chamber. .in ultrasonic generator of the type used therapeutically, such as the Siemens Sonostat 631, operating at 800 kc. with a maximum power output of 3 watts per sq. cm., was used successfully. .A diagram of the transducer and cell appears in Figure 3. .I cubical water tank, 10 cm., on a side, was constructed of Lucite and the trans'lucer was nealcd into the bottom of this bath with an epoxy resin. -A planocclncave, spherical 8%
lens of 8-cm. focal length was machined from Lucite and placed on the face of the transducer. The cell Mas constructed of Lucite pipe, 5-cm. o.d., 15 cm. long, with a flat Lucite bottom, 1 mm. in thickness. ,Ibout 10 to 30 ml. of sample solution was placed in the cell. The ultrasonic waves were focused by spherical lens onto the surface of the aqueous sample, the height of the cell above the lens being adjusted for maximum fog production. Under low power, the ultrasonic waves produce a mound of solution about 1 cm. in height above the surface of the sample liquid. As the power level is raised, the mound grows, becomes unstable, and finally bursts into a fountain spraying liquid to a height of some 15 cm. This fountain is accompanied by copious quantities of fog, produced probably by the countless tiny implosions which happen to occur exactly a t the surface of the solution. This quite stable fog is carried by the nitrogen stream into the torch chamber. Optical System. X 67-mm. focal lcngth spherical quartz lens, 30 mm.
I
\
UI trosonic Transducer
'A
Figure source
Lucite Lens
-
Ultrasonic atomizer system
in diamclter. ~ v w placcd tia1fw:iy bctwwn tho torch tip and t h r entrance slit of the monochromator. 'I'hcl distance betwccn the lcna and monochromator was 140 mm. The monochromator employed was the .Jarrell-.ish AIodel 82-000 Ebert scanning spectrophotometer with number 35-00-58-29 diffraction grating, 30,000 grooves per inch, blazed for 5000 .A. Twenty-five-micron fixed slits have been used throughout this work. The detector was an RCA 11'28 multiplier phototube, powered by a Fluke type 412.i power supply and used in a cathode follower circuit essentially that described by Whisman and Eccleston (5). Spectra and intensities were recorded on a Weston Model 670 recorder; maximum full scale sensitivity 5 mv. RESULTS
Plasma Gas. For a diatomic gas, the ability of the i)lasma to induce electronic transitions depends for the most part on the dissociation energy of
Bockground Spectrum
I
(N2+H20)
I00 A
Figure 4. Background spectrum of nitrogen saturated with water vapor, arbitrary intensity units VOL. 36, NO. 2, FEBRUARY 1964
413
Table 1.
Element
Plasma torch Detection Wavelength, A. limit, p.p.m.
A1 A1
3961,5
Au Ba Bi Ca Cd co cu Fe
2676 4554 4722.5 4227 2288 3453.5 3248 3720 2536.5 6708 2852 4031 3415 4058 2659.5 3034
...
HZ
LP Mg R.1n Xi Pb Pt Sn Sn Sr T1
v
Zn Zn Zn 0
Detection Limits of Elements
From Gilbert (5’).
Table II.
0.02 ... 0.02 0.08 2 0,005 0.1 0.01 0.001 0.015 0.2 0 0 0 0
3961.5 4842 2676 5536 4722.5 4227 3261 3530 3248 3248 2536.5 6708 2852 4031 3254 5 4058 2659.5 3034 3416 4607 3780 5469 2139 4810.5 5200
02
005 004 01 0.08 0.1
0.2
...
...
4607 5350.5 4379 2139 4810.5
0.05 0.07 0.015 0.1 0.2
...
...
loa
3a.b 5” 1.1 40” 0.08 5” 4.2 1.1 1.1 30”
0.18 1.9 0.01 0.7” 14 13” 1000 25.1
b
0.07 0; 62 10 500” . .
80”s‘
Band.
Analysis of Zinc Solutions
Concentration, p.p.m. Actual Found 400 405 40 39.8 10 9.9 100 104 200 200
the molecule. Thus, carbon monoxide would appear to offer go’od excitation temperatures but has severe chemical disadvantages. Monatomic gases such as helium may be used, but the effective “temperature” would be considerably lower. The nitrogen plasma used for the work reported in this communication appears to be the most useful readily available. The phrase “temperature of the plasma” has, in actuality, no exact meaning, since nothing remotely resembling thermal equilibrium is ever attained. The electron gas is a t a temperature of perhaps 50,000° to 100,000’ K., whereas the temperature of the ions in the plasma is about a factor of ten lower, and the average temperature of the neutral molecules is still lower. Lines of metals requiring an excitation energy of 8.5 ev. (10,000” K.) were observed with an intensity high enough to be analytically useful. The spectrum of nitrogen containing water vapor is of special importance since it represents the background interference to be expected whenever a determination of ions in aqueous solu414
Oxyhydrogen flame Detection Wavelength, A. limit, p.p,m.
ANALYTICAL CHEMISTRY
tion is to be made. Figure 4 is a rough sketch of this background under conditions of low sensitivity and fast scan. Little interference is to be expected from 2000 to 2900 A., although some low intensity bands appear. The region 3000 to 3600 A. contains nitrogen bands of very high intensity and the OH radical band, such that perhaps two thirds of this region is completely unusable. Low intensity bands appear to about 4300 but unless the line of an element to be determined lies very close to a band-head (a quite unlikely event) little difficulty with background interference is to be expected. The spectrum between 4300 and 6100 A. is quite free of bands and lines; some interference then appears to 7840 A. Base-line stability is in all regions very good. The appearance of the background spectrum will change markedly with the height above the torch tip a t which the plasma is viewed, the rate a t which the spectrum is scanned, the resolution of the monochromator, and the response time of the recorder, so that Figure 4 must be considered to be only a rough approximation. Detection Limits of the Elements. Detection limits of all elements studied thus far are listed in Table I. Only a few of the analytically useful lines are listed. Those given are, in general, the most senhitive lines which are satisfactorily free of interference. The term “detection limit” is here defined as 1% full-scale deflection on the 5-mv. recorder a t maximum sensitivity. For purposes of comparison, detection limits for the ~
hydrogen-oxygen flame are also given. These values are taken from Whisman and Eccleston ( 5 ) whenever possible, as their det’ection system, except for the use of a more sensitive recorder, is most comparable to ours. These detection limit’s do not necessarily represent the best values obtainable with this instrument. Improvement in sensit’ivity may be expected by modification of the rat,her crude ultrasonic atomization cell, by use of a high power, higher frequency ultrasonic generator, by use of larger slits when high resolution is not required, and by altering the design of the torch chamber, which in t’urn alters the shape and physical characteristics of the plasma, and by use of a more sensit’ivedetect,ion system to take advantage of the high signal to noise ratio. Detection limit,s for the more easily excitable elements such as the alkali metals might be improved by using a carrier gas which produces a “cooler” plasma. A11 data reported in Table I were taken with the nitrogen plasma operating at’ an input power of 400 wat’ts (200-ma. plate current at 2000 volts), Determination of Zinc. To ascertain the accuracy with which the instrument as described may be used for analytical determinations, five synt’hetic zinc “unknowns” were prepared and analyzed by a modified “st’andard addition” method. A 25-m1. sample of a zinc nitrate solution was pipetted into the sample cell, and atomization was begun. The monochromator was set manually a t 4810.5 and the height of the source relative to the slit, the nitrogen flow rate, the power level, the ult’rasonic atomizer, and the position of the 67-mm. lens were adjusted to give the maximum signal. These adjustments can be done quite rapidly, and need only to be approximate. The background intensity was recorded a t 4809 A. Since the background signal was constant, it needed only to be measured once, and then the monochromator was again set a t 4810.5.A4.;it was left in this position for the durat’ion of the run. approximately 2-minute intervals, 0.250 ml. of a zinc solution of k n o m concentration was injected through a serum cap stopper into the unknown solution by a hypodermic syringe. Three such additions were made, and the net emission intensity of the initial solution and that after each addition were plotted z’s. the known concentration of zinc in the solution due to the added zinc. In all cases, the resulting curve was a straight line, and the analysis was completed by dividing the net emission intensity of the unknown by the slope of the line. With no prior experience with this technique, an operator was able to complete each analysis in less than 15 minutes. The results (Table 11)
clearly indicate the analytical potentialities of the method. The error is due, for the most part, to short-term fluctuation in the rate of ultrasonic atomization. DISCUSSION A N D CONCLUSIONS
The degree to which the signal enhancement over conventional instruments is due to the ultrasonic atomization rather than to the plasma excitation cannot be answered readily because of the quite different requirements of the plasma torch 1s compared to a combustion-type flami.. The ultrasonic atomizer described, although far superior to a conventional atomizer when used with the plstsma, would probably show no great increase in sensitivity if used, for example, in the oxygen f l o ~to a hydrogen or acetylene burner. I t should be remembered that the concentration of “fog” produced by an ultrasonic generator may depend markedly on the physiical properties of the solution to be analyzed ( I ) . Some practical advantages of the nitrogen plasma in addition to its high energy, stability, and constant background level should be mentioned:
First, oxide bands seldom appear. Many of the elements in Table I tend to show quite strong oxide bands in chemical flames. I n no case were such bands observed in the nitrogen plasma. Second, metal hydroxide bands, although observable, are of low intensity and therefore do not cause the difficulties usually observed in chemical flames. Third, preliminary experiments have indicated that the sample matrix effects are much less important with this source than with chemical flames. For example, more than 100-fold excesses of aluminum and phosphate have no effect on the emission intensity of calcium and strontium; surface-active agents have essentially no effect (although large amounts of foam cannot be tolerated) ; high concentrations of hydrochloric acid and urea cause no change in the signals of zinc and strontium; and methanol up to 507, by volume does not alter the intensity of the zinc 4810.5 line which shows marked chemiluminescent enhancement in the hydrogen-air flame. Large increases in viscosity will, however, decrease the efficiency of ultrasonic atomization. A
more complete account of plasma excitation in nitrogen and other gases is in preparation. LITERATURE CITED
(1) Antonevich, J. N., “Ultrasonic atomization of liquids,” I R E Trans. Ultrasonics Engineering, PGUE-7, p. 6 (Feb.
1959).
( 2 ) Gilbert, P. T., Paper presented at the
Third Pacific Area National Meeting, American Society for Testing and Materials, San Francisco, October 1959. (3) Mavrodineanu, R., Hughes, R. C., Spectrochim. Acta 19, 1309, (1963). ( 4 ) Roddy, C., Green, B., Electronics World 6 5 , 29 (1961). ( 5 ) Whisman, bl., Eccleston, B. H., AKAL.CHEM.27, 1861 (1955). C. DAVIDWEST DAVIDN. HUME De artment of Chemistry and t i e Laboratory for Suclear Science Massachusetts Institute of Technology Cambridge 39, Mass.
RECEIVED for review August 26, 1963. Accepted Xovember 11, 1963. This work was supported in part by a predoctoral fellowship awarded to C. D. West by the Iiational Institutes of Health, and by the United States Atomic Energy Commission under Contract AT(30-1)-905.
Di- p- na pht hy It hioca rbazo ne (Dina pht hiz o ne) Compa red with Dithizone as an Analytical Reagent for the Determination of Trace Metals in Natural Waters A Preliminary Investigation SIR: The analytic11 properties of dinaphthizone (trivial name for di-bnaphthylthiocarbazone) are similar to dithizone (diphenylthiocarbazone) as shown by Hubbard et al. (1, 2, 6). An additional comparative exploration of these reagents is made with special reference to water E nalyses in this paper. For a general review on di-@naphthylthiocarbazone, and bibliography on analogous compounds, see Sandell ( 7 ) . Some of the properties of these two reagents are comrared in Table I. The observed molar absorptivities of dinaphthizone are proportional to its purity. The 67,000 estimation is probably just under the true value. Dinaphthizone looked attractive for the analysis of natural waters for heavy metals by reason of its high molar absorptivity and low acidity. Dinaphthizone’s low acidity makes it possible to extract quantitatively its metal compleses as well as uiireacted dinaphthizone from alkaline (pH 8 to 10, samples ~ i t as h little sts 1 part chloroform per 40 parts sample. This leaves
the sample relatively uncontaminated, so it can be used in subsequent analysis. This latter feature makes it look very promising for sea-water analysis, where sample size is usually limited. The emphasis on alkaline extraction follows from the observation that both dithizone and dinaphthizone transfer more metals more completely and rapidly from aqueous phase to organic phase under alkaline conditions than under acid of neutral conditions. The rapidity with which equilibrium is reached in such a transfer, aqueous to organic, is roughly proportional to the pH. The di-@-naphthythiocarbazone used in this investigation was synthesized according to the method of Hubbard and Scott (4, 6). The starting material in this synthesis is @-naphthylamine, a potent carcinogen; therefore the method of Hubbard (4)is recommended which starts with 2-naphthylhydrazine hydrochloride. At the time of writing, dinaphthizone was available from Light Chemical Co. Ltd., Colnbrook, Bucks, England. The purity of this reagent is
important; therefore one is urged to acquaint oneself with the various methods of purifying it (3, 5 ) . EXPERIMENTAL
Apparatus. A glass Millipore filtering unit was used with HA filters. The glass parts were coated with ceresin wax. For extraction 500-ml. quartz flask with ground-quartz stopper was prepared, with a teat-shaped bulge on the bottom for collecting the
Table I.
Properties of Dithizone a n d Dinaphthizone
Dithi- Dinaphthizone zone 32,000 67,000 (8)
Molar absorptivity Maximum absorption at 6200 A . 6480 A. Color of CHCL solution Green Blue green Follows Beer’s Law Yes Yes
VOL. 36, NO. 2, FEBRUARY 1964
415
