chemical yield corrections a e r e applird to thece reiults. l h t l e r and T h o m p o n ( 2 ) ha\ e hTted the Cs Talues obtained b~ qeveral laboratories for the granite mentioned in 'I'ahle 1'. The alerage of these \slue*, neighted acccrding to the number of sample< each \ alue reprebentb, is 1.9 fig per gram 'fhia result and the reco\ er? te-ts dem-ibed above are the onlj niea\ure- of acc x a c y a\ ailable at prewit LITERATURE CITED
( 1 ) Brciadbank, It. LY d., Hading, It. I )
(1960)
C.,Dhabanandana, ,
dnalyst 85, 36.5
(2) Butler, J. R., Thompson, A. J., Gcochank. Cosmochim. Acta 26, 1349 fl962). j
-
_
.
~
( 3 ) Collins, A. Gene, ANAL. CHEM.35,
1258 (1963). 14) Fabrikova. E. A., Zhur. Anal. Kham. 14. 41 1195d). (5) Finst&, H . L., Kinsley, XI. T., U. S. Atoniic Energy Comm. Rept. BNL 5334, Brookhaven Xational Lab-
oratory, Upton, 3.Y.) February 1961. ( 6 ) FiJlSimI, T. R., Scripps Institution of Oceanography, La Jolla, Calif., private communication, 1963. ( 7 ) Healy, T. Y.,Davies, B. L., U. K. Atomic Energy Researrh Establishment Kept. AERE-R-2968, Harwell, Berkshire, England, 1959. 18) Kahn, B., Smith, 11. I Straub, C. P., ANAL. CHEM.29, 1210 (1957). ( 9 ) Kelley, 11. T., Fisher, I). J., Jones, H . C . , l b i d . , 31, 178 (1959).
(10) Lundell, G. E . F., Hoffman, J. I., Outlines of Methods of., Chemical Analysis," p. 105, Wiley, Kew York,
1938. (11) LTcKenzie, T. R., Schulz, W. W. ( t o U. S. Atomic Energy Comm.), U. S. Patent 2,982,785 (May 2, 1961). (12) Prussin, S. G . , hlass. Inst. Technol. Lab. Yucl. Sci. Progress Rept., pp. 30-32, May 1, 1960. (13) Salazar, A., AIass. Inst. Technol. Lab. Sucl. Sci. Progress Rept., pp. 19-20, Xovember 1, 1959. (14) Smit, J. van R., Robb, W., Jacobs, J. J., J . Inorg. .Vucl. Chem. 12, 95, 104 (1959). RECEIVEDfor review August 21, 1963. Accepted October 18, 1963. Paper presented at ihe Midwest Symposium on Spectrosco Society for Applied Spectroscopy, Rlrcago, Ill., 1Iay 1962.
Fluorometric Determination of Lipase, Acylase, Alpha-and Gamma-Chymotrypsin and Inhibitors of These Enzymes G. G. GUILBAULT and D. N. KRAMER Defense Research Divkion, U . S. Army Chemical Research and Development laboratories, Edgewood Arsena/, Md.
b A new, rapid meihod i s described for the deteimination of steapsin, porcine pancreas, and wheat germ lipase, acylase, and chymotrypsin in the presence of othei- esterases. The method i s based on the hydrolysis of nonfluorescent fluorescein esters b y these enzymes. The rate of change in the fluorescence of the solution due to production of fluorescein, hF/At, i s measured and correlated with enzyme activity. The preparation of the substrate solutions is rldatively simple, and analysis requires only 3 to 5 minutes. Employing the method described, 0 . 0 1 2 5 to 0 . 2 5 0 mg. per ml. of wheat germ or steapsin lipase, 0.00600 to 0.100 mg. per ml. of acylase, 0.0025 to 0.0500 mg. per ml. of porcine pancreas lipase, and 0.167 to 1 .30 mg. per mi. of a- or y-chymotrypsin may b e determined with a relative standard deviation of about &1.5 to 2.0y7,. Two minutes incubation permits the determination of 0.0330 to 0.330 kg. per ml. of sarin or Systox with a relathe standard deFinally, viation of about 1 2 . 0 % . 0.070 to 0.50% solutions of Triton X-100 were analyzed with a relative standard deviation of about =t1 .A?&.
A N T U F T H E MLIETHODSfor
enzyme analJ,sis used today are undesirahle lmause of the long and tedious procedures involvcd. This is especially true in the rase of lipase> analysis. which require* the action of enzyme powder on neutral olive oil for either 30 minutes or 2 hours at elevaterl temperatures,
with the assay performed either by manometric ( I ) or t,itrimetric procedures (8,I O ) . Greenstein (4) and Laskowski ('7) have recently reviewed the current procedures for acylase and the chymotrypsins, respectively. The most sensitive methods for organophosphorus compounds, such as sarin or Systos involve an enzymatic method, the reaction being followed either electrochemically ( 5 ) or by observing the change in the pH of the solution (S). Finally, titrimetric methods for water soluble substrates, such as Tween 20, are given in Rier's "Methods in Enzymology" ( 2 ) . In a recent scientific communication ( 6 ) , a simple, rapid procedure was described for t'he assay of lipase activity in the presence of other esterases. The method was based on the hydrolysis of fluorescein esters catalyzed by lipase. The rate of change in the fluorescence of the solution, due to production of fluorescein, was measured and correlated with enzyme activity. The present study discusses the esperimental parameters involved in this technique and extends the method t o the determination of porcine pancreas and wheat germ lipase, to acylase, and to CY- and y-chymotrypsin. Finally, various organophosphorus compounds, such as sarin or Systos, are determined in submicrogram amounts by their inhibition of liliase activity. EXPERIMENTAL
ENZYMES. Lipase, porcine pancreas, Calbiochem Co.; activity, 2.1 JYilson units per mg. Reagents.
Aiunit represents 0.05 meq. of titratable fatty acid formed from the action of enzyme powder on 1.0 ml. of neutral olive oil in 30 minutes a t 37" C., as assayed by the procedure of Lazo-JJ7asem (8). Lipase, steapsin, Xutritional Uiochemical Co.; activity, 0.761 units per "9. Lipase, wheat germ, Calbiochem Co.; activity, 50 fil. of COz liberated in 30 minutes from triacetin by the action of 1 mg. of enzyme a t 37' C. .\cylase, purified enzyme obtained from the rational Institutes of Health ( 4 ) ; activity per milligram, 0 . 7 0 units (by comparison t o the activity of steapsin lipase, using dibutyrj-lfluorescein as substrate). Chymotrypsin, CY- and y-, Calbiochem Co., had activities of 7.8 units per mg. (Kunitz) and 6000 .\TEE unit$ per nig., respectively. SUBSTRATES.Diacetyl- and dibutyrylfluorescein solutions, 5 X l0-5MJ were prepared by dissolvinp the appropriate amount of material ( 6 ) in 5 ml. of Alethylcellosolve and 95 ml. of tris buffer, pH 8.0. TRIS BUFFER. Tris(hydrorymethy1) aminomethane, pH 8.0, O.l.lf, was y e pared by dissolving the appropriate amount of Sigma 7-9 buffer (Sigma Chemical C0.j in distilled water. HC1, O . l S , was added to adjust the p H to 8.0. ISOPROPYLMETHYL PHOSPHONOFLUORIDATE (SARIN). Aqueous solutions were obtained by dissolving pure sarin, prepared in these laboratories, in triply distilled water. 0 , O - DIETHYL 0 - 2 - (ETHYI,THIO)ETHYL PHOSPHOROTHIOKATE (Swi-ox), Solutions of pure Systos. containing either the p-o isomer, or a mixture of isomers (50150) (Chemapro Co.), were ],repared in tris buffer, pH 8.0. VOL. 36, NO. 2, FEBRUARY 1964
409
ALKYL PHENOXY POLYETHOXY ETH(TRITONX-100) (Rohm and Haas Co.). Amaratus. d Klett fluorimeter. equ;iped with a General Electric -%H 4 mercury vapor lamp as the energy source and a Rubicon galvinometer (sensitivity 0.005 pa. per mm.), was used to assay enzyme activity. .%Iternatively, an .Lminco-Bowman spectrofluorometer, equipped with a Xenon lamp, an optical unit for proper control of the fluorescence excitation and emission wavelengths, a photomultiplier microphotometer, and a Beckman linear recorder, were used. Since the maximum excitation wavelength for fluorescein is 470 mp, and the optimum emission wavelength is 510 mp, these wavelengths should be used for maximum sensitivity. Procedure. ENZYME - ~ C T I V I T Y . Twenty milliliters of a 5 X 10-5M solution of dibutyrylfluorescein (or a 5 x 10-h.V solution of diacetylfluorescein for assay of wheat germ lipase) is placed in a 25-ml. fluorescence cell in the Klett fluorimeter, and the fluorescence is adjusted to read zero. At zero time, 1.0 ml. of a solution of t'he enzyme to be assayed (containing 0.25 to 5,O mg. of st,eapsin or wheat, germ lipase, 0.05 to 1.0 mg. of porcine pancreas lipase, 0.120 to 2.0 mg. of acylase, or 3 to 25 mg. of a- or 7-chymot'rypsin) is added, and the change in the fluorescence of the solution, due to hydrolysis of the substrate, is then recorded us. time, usually for a period of two minutes. ANOL
lipase
Dibutyrylfluorescein + HzO ----+ p H 8.0 fluorescein + 2 butyric acid The slope of this curve, A F / A t , is determined, and from linear calibration
Table I. Effect of Substrate Concentration AF/At,
diRuF, M
units/ min.
x 10-4 x 10-6 1 x 10-6 5 x 10-6 1 x 10-6
100 90 70 50 35
1 5
Table II.
Effect of pH
A. Effect of pH on the fluorescence Fluorescence, PH Klett units 115 6.76 119 7.0 123 7.5 128 8.0 121 9.0
B. Effect of pH on the rate of enzyme catalyzed hydrolysis
410
&Flat,
PH
units/min.
5.70 6.76 7.0 8.0 9.0
43 57 70 65 50
ANALYTICAL CHEMISTRY
cein esters prepared-the diacetyl, dipropionyl, dichloropropionyl, dibutyryl, divaleryl, and dicaproyl-were 1.5 colorless; the eosin esters-diacetyl, dipropionyl, and dibutyryl-were pale cream colored. The kinetics of the reaction of all the substrates Rith six different enzymes were elucidated and were found to obey JIichaelis-Xlenten kinetics. These results will be published in a future article. For all enzymes, except porcine pancreas, the rate of hydrolysis of the fluorescein and eosin esters decreased in 20 40 60 80 100 the order: acetyl > propionyl > FLUORfSCfNCf butyryl > valeryl > caproyl. K i t h porcine pancreas, the divaleryl and Figure 1. Fluorescence-time curve for dicaproyl esters of fluorescein had a the enzymatic hydrolysis of 5 X 1 O-jM higher rate of hydrolysis that the didibutyrylfluorescein b y 0.01 70 unit of butyryl ester. The eosin esters lipase hydrolyzed very slowly, upon treatment with lipase, even a t p H 10.0, and hence were not considered as possible subplots of A F l A t us. enzyme concentration, the activity of the unknown strates. enzyme may be calculated. Since the spontaneous rates of ['sing the Iminco-Howman spectrohydrolysis of the diacety1 and diprofluorometer, 2.0 ml. of substrate solution pionyl esters were so much greater than is used, and a t zero time 0.1 ml. of the the dibutyryl (which had a neghgible enzyme solution, containing one tenth rate of spontaneous hydrolysis for of the amounts described meviouslv, is which no correction is necessary), the added. dibutyryl fluorescein was chosen as the The enzyme solution must be prepared fresh before each set of runs to obtain substrate of choice for analysis of constant, reproducible results. The steapsin and porcine pancreas lipase, calibration plots need not be repeated acylase, and chymotrypsin. Since the daily, provided they are initially derate of hydrolysis of the dibutyryl ester termined with fresh enzyme. by wheat germ lipase was slow enough I n assay of wheat germ lipase, a corto introduce error in the measurement of rection must be made for the spontanethe rates, expressed as AF At, the ous hydrolysis of the diacetylfluorescein. diacetyl ester was used in all assays of This is done by subtracting the rate of this enzyme. Honever, a correction for nonenzymatic hydrolysis from the value of AF/At obtained. spontaneous hydrolysis must be made. DETERMINATION OF ORGANOPHOS- The effect of substrate concentration PHORUS COMPOUXDS. h solution (0.05 on the rate of hydrolysis is shown in ml.) containing 0.08 to 1.0 pg. of Systox Table I. Although the rate is slightly or sarin is incubated for two minutes higher using a 10-4Alfsubstrate solution, with 0.05 ml. of a 1 mg. per ml. steapsin appreciable solubility problems eyist solution in a I-em. fluorescence cell which affect the reproducibility. A (Pyrocell Manufacturing Co.). At the 5 X l O - s J f solution may be prepared by end of this period, 3 ml. of a 5 X I O + X first preparing a stock l O - 3 J f solution dibutyrylfluorescein solution is added, and the slope of the resulting curve, in ;\lethylcellosolve, then diluting 5 ml. AF,lAt, is obtained. From linear caliof this solution to 100 ml. with tris bration plots of AF/'At us. inhibitor conbuffer. The resulting solution is slightly centration, the amount of unknown cloudy, and represents the limit of material may be determined. solubility that can be achieved for DETERMINATION OF TRITON X-100. maximum reproducibility. Although To 2.0 ml. of a 5 X 10-5.1f solution of more concentrated solutions might be dibutyrylfluorescein is added 0.1 ml. prepared by addition of more Methylof Triton X-100 (containing 1 to l0Yo cellosolve, quantities of this soIvent Triton by volume), and the fluorescence is adjusted to read zero. + i tzero time, >5%, adversely affect the repro0.1 ml. of a 1 mg. per ml. steapsin ducibility. Experimentally, the 5 x solution is added to effect the hydrolysis l O - s X solutions of substrate, though of the ester, inhibited by Triton. From slightly cloudy, could always be adcalibration plots of A F / A t us. 1/ [Triton], justed to zero fluorescence with little the amount of unknown Triton may be difficulty. calculated. Effect of Enzyme. I n addition to the lipases, acylase and chymotrypsin DISCUSSION also effect the hydrolysis of the fluorescein esters, and hence are deEffect of Substrate. A compreterminable by this procedure. The hensive series of fluorescein and eosin a- and y-chymotrl-psins hydrolyze diesters were prcparcd for testing as butyrylfluorescein at apl)ro\imately the substrates, t o obtain the optimum same rate, but the @-chymotrypsin acts conditions for analvsis. The fluores-
/ 1 1 1 1 1 .
very slowly, possessing a rate '/z to that of the a- or y-compounds, and hence is easily identified (assuming that pure samples of the enzyme forms were obtainable). The chymotrypsins may be differentiatfed from the lipase and acylase since high concentrations of these enzymes are needed to effect the hydrolysis of the substrate. The presence of acylase niay be determined by the use of suitatile inhibitors that attack acylase and not lipase. Other hydrolases such as hcrse serum cholinesterase (-4rmour and Co.) or acetylcholinesterase (JTintlirop Labs), penicillinase (Baltimore Biological Labs) or bovine plasma dbumin (Armour Labs), do not hydrolyze the substrate, and do not int.erfere. Effect of pH and Temperature. Fluorescein exhibits a maximum fluorescence a t about pEi 8.0, and t h e rate of hydrolysis of dikiutyrylfluorescein reaches a maximum a t between pH 7.0 and 8.0 (Table I [ ) . The greatest sensitivity and reproducibility can be achieved a t a pH of 8.0 in tris buffer. The rate of hydrolysis of substrate by enzyme depends uport the temperature used. Hence, for best results, all determinations and calibra-.ion plots must be run at, the same temlxrature. I n these experiments t,he temperature was regulated to xithin + I c C. either by a constant temperature room or bath. At higher temperatures (30" to 40" C.) and higher pH's ( 8 . 5 ) considerable spontaneous hydro1 occurs, adversely affecting the accuracy and reproducibility. .It 20" to 30" C. and a pH of 8.40, very little spontaneous hydrolysis occurs (AFl At = 0.44). Stability of Reagents. The enzyme and substr:ttt solutions arc :iormally stored in a refrigerator a t 8" C. when not in usc. Thus, these reagents may he used for se-m;eral days rrith good rtproducihility. ;\fttr 4 to 6 hours without refrigeration. considerablr hydrolysis of the fluorewein ester solutions occur. For best results, it is rccommcnded t h a t a stock solution of substrate, 10-3.11, be prepared and dilutions made every 2 to 3 days, and that the enzyme solutions be prepared fresh daily. .iddition of a few milligrams of bovine plasma albumin stabilized the enzyme. Effect of Incubation. I t has been reported (9) that lipaises are normally insensitive to organoluhosphorus compounds, and thus can be differentiated from the cholincster,Lses which are affectcd by submicrogram amounts of these inhibitors, Howcver. it was found in these studies that both sarin and Systos in concentrations as low as 0.033 p g . per ml. of solution can be determined after 2 minutw incubation with steapsin lipase. The action of these inhibitors on vwious enzymes is shown in Table 111. n.
Table 111.
diBuF
diBuF
=
Effect of Inhibitors
A. Effect of time of incubation on the extent of inhibition 5 X 10-6M; steapsin = 0.025 mg. per ml.; sarin = 0.033 pg. per ml. Time of AF/At, incubation min. units/min. 0.0 60 2.0 30 5.0 30 10.0 30
B. Effect on various enzymes enzyme = 0.025 mg. per ml. of steapsin or wheat germ (WC) or 0,0052 mg. per ml. of porcine pancreas (PP). Incubation = 2 minutes A F / A t , units/minute Inhibitor concn., pg. per ml. Sarin Systox Steapsin PP WG 0.0 0.0 31 41 30 0.170 0.0 16 2 2 0.0 31 21 0.012 18 41 0.020 15 0.0 0 10 0.70 0.0 =
5 X IO-Sill;
Table IV.
Determination of Enzymes
Steapsin, mg./ml. Rel. error, Added Found" 76 0 0 0 0250 0 0250 +2 0 0 0.500 0 0510 +0 0 0 0625 0 0631 -1 6 0 125 0 123 +o 4 0 250 0 251 Re1 std. dev f 1 6'33 Wheat germ, mg./ml. Rel. error, Added Foundn c/c 0.0124 -0.8 0.0125 0.0251 +0.4 0.0250 0.0510 +2.0 0.0500 0.101 +1.0 0.100 0 199 -0.5 0.200 Rel. std. dev. 4 ~ 1 . 5 7 , a-Chymotrypsin, mg./ml. Rei. error, Added Founda % $1 8 0 167 0 170 -1 0 0 327 0 330 +1 0 0 660 0 667 +l 6 1 20 1 22 Re1 std dev f 2 5 ' 3 a
Porcine Pancreas, mg./ml. Rel. error, GAdded Founda /C 0 00250 0 00253 +12 0 00500 0 00506 +1 2 0 0100 0 0100 0 0 0 0250 0 0250 +o 4 0 0500 0 0491 -1 8 Re1 std dev 5 5 Acylase, mg./nil. Rel. error, C' Added Founda 'c 0.00600 0.00610 +1.6 0.0125 0.0125 0.0 0.0250 0.0251 $0 4 0.0500 0,0490 -2.0 0.100 0.101 +1.0 Rel. std. dev. = t t l $)? r-Chymotrypsin, mg./ml. ~ Added FnundR 0 170 0 172 0 340 0 340 0 680 0 687 1 30 1 28 Re1 std dev
c0
+I
2
0 0 +I 0
-1 5 &I 5 5
Each value represents an average of 3 or more determinations
Table V.
Determination of Inhibitors
Sarin, pg /nil Re1 error, C/I Added Founda +o 0 0 0330 0 0333 0 0568 -0 4 0 0570 -1 8 0 114 0 112 0 0 0 170 0 170 0 317 +I 3 0 321 Re1 std dev f l 5 7 ,
Systox, pg
Added 0 0660 0 132 0 198 0 264 0 330
Triton X-100, Vo in overall Soln. ildded Founda 0 0700 0 0701 0 100 0 100 0 173 0.175 0 2.50 0 253 0 375 0 374 0 500 0 501
Q
~ error, 1 ,
/ml Re1 error, rFound0 0661 +I 3 0 130 -I 5 0 108 0 0 0 260 -I 5 0 333 +I 0 Re1 std dev f:!Oyc Rel. error, c/1 +1 4 0 0
-1 +I
1
2 -0 3 +2 0 Re1 std dev & I 4%
Each value represents an average of 3 or more determinations.
VOL. 36, NO. 2, FEBRUARY 1964
41 1
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 #
FluoriLaeometric Wasem procedure (8) 482.5 716.0 2100
500
710 2110
124142
3323
3220
122005
2440
2450
Lipase, Pancreatic, Wilson Lot #
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
