For quantitative determination of diaphorase and resazurin, only the latter part of the coupled reaction was usedresazurin diaphorase. i.e., S A D H At a NADH concentration of 6.7 X 10-+3X,the rate of production of resorufin, AF per minute, is proportional to the concentration of diaphorase and resazurin with good accuracy and reproducibility (Table I I). At diaphorase and resazurin concentrations of 0.08 unit and 6.7 x 10-6M, the rate of reaction is proportional only to the concentration of NADH. More data on the kinetics of this reaction can be found in another publication(5). The application of this procedure to the determination of other dehydrogenases should be possible. Any enzymic reaction capable of reducing KAD + or KADP+ to NADH or KADPH should be measurable in this system. Stability of Reagents. T h e stock resazurin solution, 2 X 10-4X in methyl cellosolve, is stable for a t least a
+
+
year, and the NA4D+and NADH solutions are stable for a week when stored at 5' C. The substrate solutions are stable for months a t room temperature. The only unstable reagent is diaphorase, and solutions must be prepared fresh each day. The calibration plots, however, need not be repeated daily, provided they are initially determined with a n enzyme solution that was freshly prepared. Effect of pH and Substrate Concentration. The rate of reaction is proportional t o t h e concentration of substrate used-Le., sodium lactate, etc.a t low concentrations. Generally, t h e rate becomes independent of t h e concentration of substrate a t concentrations > 1 O - * M , and this maximum concentration was used in the analysis (see Reagents for the concentration of each substrate used). Since the rate of all these reactions is higher a t pH's of 8-9 (the reverse reaction becoming predominant a t lower pH's), and since
the fluorescence of resorufin is also optimum a t p H 8-9 (S), this approximate range was used in all analyses. The p H optimum for each reaction is given in the Reagents section under the individual substrate. LITERATURE CITED
(1) Guilbault, G. G., Kramer, D. N.,
ANAL.CHEM.36,409 (1964). ( 2 ) Zhid., p. 2497. ( 3 ) Guilbault, G. G., Kramer, D. N., Zhid., 37,120 (1965). ( 4 ) Guilbault, G. G., Kramer, D. N.,
Edgewood Arsenal, Md., unpublished data, 1965. ( 5 ) Guilbault, G. G., Kramer, D. N., Goldberg, P., J . Phys. Chem., in press. ( 6 ) Kramer, D. N., Guilbault, G. G., ANAL.CHEM. 36. 1662 (1964). - - , (7) Lowry, 0. H.,'Roberts, N. R., Chang, M . , J . Biol. Chem. 222,97 (1956). (8) Lowry, 0. H., Roberts, N. R., Kapphahn, J. I., Zhid., 224, 1047 (1957). RECEIVEDfor review April 21, 1965. Accepted July 7, 1965. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 1965. \
A Constriction Response Analyzer and Its Application to Continuous Uranium Hexafluoride Determination C. W. WEBER
and W. S. PAPPAS
Technical Division, Oak Ridge Gaseous Diffusion Plant, Union Carbide Corp., Nuclear Division, Oak Ridge, Tenn. A continuous nondestructive analyzer which responds pneumatically to changes in density and viscosity of the fluid passing sequentially through an orifice and a capillary has been developed. It provides a pneumatic signal directly applicable to stream analysis and to feedback control systems. Good agreement with chemical analyses was obtained where applied to the determination of uranium hexafluoride in complex, corrosive, gas mixtures. A sensitivity of 0.03 mole % uranium hexafluoride in nitrogen was achieved, with a standard deviation of =t0.03 mole %. Little maintenance was required during a field application period exceeding three years.
C
and automatic control of the composition of gaseous and liquid systems are often required for efficient operation of modern chemical plants. This report describes a simple, low cost analyzer, responding to density and viscosity, that may fulfill some of those needs. The analyzer was designed especially for the continuous measurement of uranium hexafluoride in gas mixtures; its principles are applicable to any fluid system where the system value, density ONTINUOUS INDICATION
divided by the square of viscosity, varies with the concentration of the component sought. The authors recently reported another instrument for determining uranium hexafluoride, by the condensation pressure method ( 7 ) . While that analyzer was more specific for uranium hexafluoride, as applied to selected gas streams, the present constriction response approach has advantages: it responds more rapidly to marked, rapid shifts in uranium hexafluoride concentration; it handles a broader concentration range in a single instrument; since i t is based on different principles and does not require condensation of the constituent sought, its versatility of application is potentially greater; the instrument can be calibrated for any regions of sample pressure or flow, within the capabilities of the system components. THEORETICAL CONSlDERATlONS
If a constant flow of gas a t constant temperature and constant inlet pressure is passed through a n orifice which is operated noncritically, the pressure drop, AP, across the orifice varies with molecular weight ( 2 ) . I n the case where the orifice is operated critically, the forepressure will vary with the square root of the Gas Analysis.
rAP'l IAP1 p2 CAPILLARY
Pf
I ORIFICE
Figure 1. Series flow through a capillary and an orifice
molecular weight. If the flow passes through a capillary, selected to permit laminar flow, its pressure drop, AP', will be proportional to viscosity ( 2 ) . The relationships above indicate the responses that may be expected for a capillary or an orifice, independently applied when the flow is regulated by some device not affected by viscosity or molecular weight; volume displacement systems could be used for this purpose. For many analytical problems, however, a simplification is achieved by placing a capillary and an orifice in series (see Figure 1). The flow may be controlled by maintaining constant pressure conditions at one of the constrictions; the variable pressure at the other constriction serves as the analytical response. Actual flow may vary with concentration; but since identical flows pass through both restrictions, algebraic analysis reveals a useful relationship. For a flow restriction, at constant temperature, volumetric flow ( 2 ) is theoretically proportional to VOL. 37, NO. 10, SEPTEMBER 1965
1221
-dpTp(for noncritical orifice flow)
d@
p/
417
[for sonic (critical) orifice flow] (2)
Liquid Analysis. A similar mathematical treatment can be used for liquid systems at constant temperatures, since the flow equations for gases and liquids are similar (2). With the capillary differential pressure controlled, the analytical response AP at a n orifice is
AP:
[for viscous (laminar) flow through a capillary]
(3) where
dl PI AP AP’
D
;-q2
where D = liquid density. With the orifice differential pressure controlled, the response AP’ a t a capillary is
= molecular weight of gas = orifice forepressure = pressure drop across an orifice
o
Since the density of liquids is practically independent of pressure, no total pressure control is necessary.
If the same flow passes in series through a noncritical orifice and a capillary, the flows are equated so that the above ratios (1) and (3) become proportional to one another
I n selecting an instrument design to analyze specific mixtures, the viscosities and molecular weights (or densities) of the constituents, and the choices permitted by the working expressions, including Equations 5 through 9 for gases (or 10 and 11 for liquids), are first considered. This is illustrated for the determination of uranium hexafluoride in gaseous mixtures with nitrogen, oxygen, fluorine, and small amounts of hydrogen fluoride. \’iscosities and molecular weights of these gases are presented in Table I. Since hydrogen fluoride concentration is low, the viscosity will not vary greatly with variations in content; however, large variations in average molecular weight can occur because of changes in uranium hexafluoride concentration. The other gases tend to approximate a single gas in a binary mixture with uranium hexafluoride. Calculated theoretical responses for the components of the binary mixture of interest will indicate the approximate sensitivities to expect from various designs. Table I shows those based on Equations 5 and 7. Assuming ideal mixtures, response curves can be estimated prior to experimentation. I n terms of relative sen-
PI Pz 4
Equation 4 may be simplified by assuming either AP or AP’ to be the variable analytical pressure, and all other pressures are maintained constant or large compared to the pressure drops. Then
AP
=
K ‘M
BZ
(5)
or where the K’s are proportionality constants. For the same flow passing in series through a combination of a capillary and a critical orifice, a similar treatment results in
20
40
o
METHOD OF APPLICATION
Figure 2. designs
80
60
MOLE % UFd IN
pressure drop across a capillary = capillary inlet pressure = capillary outlet pressure = viscosity of gas =
IO
IuW
NZ
Response curves of several
sitivity (defined by lowest signal = I), the curves for Equations 5 through 9 were calculated for uranium hexfluoride-nitrogen mixtures and plotted in Figure 2 to illustrate the choices available. The combination capillary and noncritical orifice, Equation 5 and curve 5 , gives a signal linearly related to uranium hexafluoride content and has a high overall sensitivity. The lower curves in Figure 2 represent the square roots of the upper curves and, therefore, correspond to less sensitive analysis. Curve 9 is more sensitive than curve 5 above their intersection point and less sensitive below; average overall sensitivities of the two curves are equal since they are simply reciprocal relationships. The comparative sensitivities of two system choices can be altered by adjusting one of the fixed pressures in a system; this mill shift one or both of the corresponding curves. For example, if the zero concentration intercept of curve 5 is shifted to equal the zero intercept of curve 9, by increasing the capillary pressure drop of system 5 , curve 5 then becomes more sensitive than curve 9 a t all points above zero concentration. Since curve 6, or 8, is the reciprocal of curve 7, they behave, relative to one another, in a similar manner as curves 9 and 5. Departure from linearity made
or Cpon examining Figure 1 and considering the fundamental relationships (1, 2, and 3) other working expressions can be derived. For a series combination capillary and noncritical orifice, for example, both AP and AP’ could be controlled constant. Then the system pressure Pp (Figure 1) could be the analytical response and, if large compared to the pressure drops,
Pz 1222
0
=
K V T~ M
ANALYTICAL CHEMISTRY
(9)
Table 1.
Comparison of Viscosity and Molecular Weight of Uranium Hexafluoride and Constituent Gases Dr Expected relative responses at orifice
viscosity
(75” C:),
micropoises Gas (IO) Component I UFO 198 Nz Component I1 02 I
196 226
M, molecular weight
Noncritical AP= _M _ K’ o2
352
8 9 . 8 X lo-‘
D 9 . 4 8 X IO-*
28 32
7.29 6.27
x x
2 . 7 0 X 10-2 2.50 X
10-4
10-4
Critical
pt K’/’
dz
L A P
-1
VARIABLE
Figure 3.
Pressure and flow control
APT Absolute pressure transmitter DPT Differential pressure transmitter PCV Pressure control valve
curves 6, 7 , 8, and 9 unattractive for analysis over a wide concentration range. The Constriction Response Analyzer, exploiting the above considerations, was developed through several assemblies designed t o test theory in the laboratory and application in the field. Because of its linearity and sensitivity, the combination capillary and noncritical orifice, Equation 5, was selected for the determination of uranium hexafluoride in nitrogen (or with the mixture indicated in Table I). A different choice might be made for a different binary gas system-e.g., one in which the viscosity could vary considerably as concentrations vary. PNEUMATIC CONTROL COMPONENTS
While manual valve adjustment is possible for maintaining the constant pressure parameters, such an elementary system would be expensive and unsatisfactory for continuous process analysis and control, because of lag and error, Fortunately, commercially available components can perform the functions of continuously controlling or measuring pressure. Additionally they simplify, by their pressure transmitting actions, the isolation of corrosive gas systems. The Constriction Response Analyzer uses pressure transmitters that sense the pertinent pressures or differential pressures and adjust control valves accordingly, as illustrated in Figure 3. The analytical response is sensed by another pressure transmitter, the signal from which can conveniently control a process parameter, or be recorded, or simply be indicated on a gauge. Pressure Transmitter. Absolute pressures and differential pressures are measured with mechanical transmitters (Taylor, Models 206-RA and 206-RD). These are high sensitivity, balanced-beam devices (4) which deliver air pressure signals, usually amplified, in proportion to the sensed pressures. They are resistant to corrosive fluoride gases. Control Valve. Pressures are controlled with diaphragm type control valves (Hoke: P a r t No. 1148), usu-
ally with 0.025-inch ports. The valve is incorporated into a feedback loop with a pressure transmitter and is actuated by the pneumatic unbalance between the transmitter output signal and a regulated air pressure applied to the other side of the diaphragm. It is applicable to corrosive fluoride gases (9) for control of flows and pressures. LABORATORY EVALUATION WITH URANIUM HEXAFLUORIDE
Several arrangments of sequential flow through a capillary and an orifice were selected for evaluation, to determine conformance with calculated signal output for the uranium hexafluoride-air system. These include measuring the analytical signal as: the pressure drop across a noncritical orifice, Equation 5; the pressure drop across a capillary, Equation 6; and the forepressure before a critical orifice, Equation 7 . The arrangement of constant differential pressure a t both constrictions, Equation 9, was tested only qualitatively, since a nonlinear signal with low sensitivity in the high uranium hexafluoride concentration range would be expected. A fifth arrangement was experimentally
evaluated because of its simplicity; it consisted of a capillary and orifice in sequence, with flow from and into constant pressure systems. For each of the experimental arrangements, a plot of analytical signal us. concentration was calculated for comparision with the experimental values. The calculations assume ideal gases, and no viscous losses a t the imperfect orifices or turbulent losses a t the capillaries. The calculated theoretical plots, in Figure 4,use the experimental values a t 0% uranium hexafluoride concentration as datum points for determining equation constants and, therefore, indicate only approximate shape, not absolute values. Since the temperature effects are different a t the two restrictions (@, the instrument should be calibrated a t the same temperature a t which it is to be used. The laboratory assemblies were mounted in a chamber at 90' i:3" C. to avoid uranium hexafluoride condensation. Exposed system metal components were of copper, nickel, or Monel to resist the corrosive gases tested; fluorothene valve disks were used. Noncritical Orifice Differential Pressure Response, Equation 5 . The analyzer of Figure 4 was tested at two inlet pressures. The suppressed response a t the orifice, a t 9 p.s.i.a. inlet pressure, may be partially explained by the effect of density upon the orifice coefficient in this region of mass flow, as was indicated by estimated Reynolds number values (3). The flow through the analyzer was in the range of 20 to 200 standard cc./min. The response may also be affected by excessive Reynolds number a t the capillary, which would provide the equivalent of an increase in gas viscosity, reducing the sample flow. Smal
40 in. Long
Figure 4.
Noncritical orifice type analyzer
--Experimental
- - - - Calculated V O L 37, NO. 10, SEPTEMBER 1965
1223
SAMPLE
Table II. Precision of High Precision Model Constriction Response Analyzer
Gas pair
Concn. range, mole 70
COrargon Nz-CC1, Helium-air Nz-UFs
0-100 0-15 CCl, 0-20 Air 0-20 CFs
Std. dev. mole 70, absolute
ANALYTICAL
PRESSURE TRANSMITIER RANGE 2-7/8 3-1/8 PSlA
AP TRANSMITTER RANGE 0 1/8 PSI
-
-
0.15 0.05 0.05 0.03
L
AP TRANSMITTER RANGE 3/32 7/32 PSI
-
metal capillaries may depart from viscous flow (6) a t Reynolds numbers below 100. X standard deviation of +0.15 mole % was obtained a t 38% uranium hexafluoride, operating a t an inlet pressure of 9 p.s.i.a. Capillary Differential Pressure Response, Equation 6. The responses for the capillary, in series with a noncritical orifice, were nonlinear and t h e sensitivity was approximately in agreement with Equation 6 and curve 6, Figure 2. Critical Orifice Forepressure Response, Equation 7. I n this system, flow was controlled in a manner similar to t h a t in t h e noncritical type instrument, and the orifice was located downstream of t h e flow control valve. Pressure downstream of the orifice was maintained a t less than 0.1 p.s.i.a. by t h e vacuum system, and the orifice forepressure served as the analytical signal. The calibration curve is not linear and relative sensitivity is less than in Figure 4, consistent with the relationships shown in Figure 2. System Pressure Response, Equation 9. With t h e inlet flow adjusted (by feedback control) t o maintain t h e capillary pressure drop constant, and outlet flow adjusted to maintain the orifice pressure drop constant, the system pressure responded qualitatively as indicated by Equation 9. Rehponse was slow, however, at flow rates obtained with the restrictions employed in the other systems. Exchanging the control functions of the capillary and orifice, resulted in a "runaway" system. For given settings of the differential pressure controls, the system pressure increased when the starting system pressure was above a certain value and decreased when the starting pressure was below this value. This type of action would perhaps be useful in some control applications. Immediate Pressure ResponseWith Constant Inlet and Outlet Pressures. A simple intermediate pressure analyzer, with t h e capillary (0.03-inch i.d. X 40 inches) and a 0.016-inch ncncritical orifice in series, was tested. The inlet and outlet pressures were maintained constant a t 9.0 and 8.75 1i.s.i.a. The forepressure a t the noncritical orifice provided a nonlinear analytical response. 1224 *
ANALYTICAL CHEMISTRY
2 PEN RECORDER
ORIFICE
ORIFICE 0.025'' DIA.'
-
/
P
TRANSMITTER RANGE CAPILLARY .051" I.D., 15" LONG 0.5 LITER SURGE TANK OUTPUT
PCV AIR
Figure 5.
4.
TO LOW PRESSURE SYSTEM
Constriction response analyzer, improved model
If the orifice is operated critically, the downstream pressure control is not necessary; for application to noncorrosive systems where a conventional vacuum pump can be used directly, this modification could result in a substantial reduction of the initial cost. If the system to be analyzed is a t constant pressure, the analyzer would consist simply of a capillary in series with an orifice, with a pressure gauge to monitor the intermediate pressure. High sensitivities should result where large total pressure drops across the system are possible. FIELD APPLICATION TO CONTINUOUS URANIUM HEXAFLUORIDE DETERMINATION
Several models of the noncritical orifice analyzer have been applied to the continuous analysis of uranium hexafluoride in streams consisting of the gases shown in Table I. * i s predicted from flow equations and confirmed experimentally, it was the most sen4tive for the uranium hexafluoride-air system and furnished a nearly linear response to uranium hexafluoride concentration. X high precision, four-range field instrument is presented schematically in Figure 5 . The instrument case mas insulated and the temperature was controlled a t 38" i. 0.2" C. Positioning of the orifice near the gas inlet reduces the displacement lag to less than 1.5 seconds; exchanging the relative position of the orifice and capillary had very little effect on the sensitivity. Analytical sensitivity was improved by the use of a suppressed range transmitter. To dampen out small oscillations in the flow control system, a surge tank was installed directly ahead of the exit control valve, and the exit valve was modified by using a larger port (0.160inch diameter instead of 0.026 inch). Installation of two orifices as indi-
cated in Figure 5, with two differential transmitters, one having a suppressed range, results in an analyzer with four ranges, the most sensitive of which can monitor changes as small as ~k0.03 mole % uranium hexafluoride in nitrogen. The calibration curve for uranium hexafluoride was established by plotting instrument responses against analyses obtained by the alkaline peroxide colorimetric method (8). Performance. The improved field model has been operating continuously on a plant stream for more than 3 years with less than 10 hours required maintenance. The response to uranium hexafluoride concentration fluctuations is almost identical to t h a t of a more expensive .Icoustic Gas Analyzer (1) installed permanently a t the test location. The standard deviation is +0.03 mole % uranium hexafluoride in nitrogen. OTHER APPLICATIONS
Gases. The precision of measurement, determined on the high precision, four-range analyzer for several inert gas pairs, is included in Table 11. Analysis of carbon dioxide-argon mixtures is based primarily on viscosity differences, since molecular weights are close. Carbon monoxide and nitrogen give identical responses as expected, since their molecular weights are the same and their viscosities are extremely close (both 184 micropoises at 20" C.). The analyzer principles can be applied to determining viscosities of gases of known densities (6). Liquids. With liquids a simpler instrument is possible since there are only two pressure variables, E q u s tions 10 and 11. Tests with acetone~
water mixtures confirmed t h e applicability of the principles to liquid analysis. LITERATURE CITED
(1) Bogardus, B.J., Ritter, R. C., “ACOUS-
tic Gas Analyzer Development and Manufacture,” Union Carbide Nuclear Co., Oak Ridge Gaseous Diffusion Plant, February 4, 1959 (K-1240). ( 2 ) “Chemical Engineers Handbook,” John H. Perry, Ed., 3rd ed., pp. 387 and 403, RIcGraw-Hill, New York, 1950.
(3) 1bid.JP. 405. (4) Fribance, A. E.,“Industrial Instrumentation Fundamentals,” p. 218, fig. 9-38, McGraw-Hill, New York, 1962. ( 5 ) Lunge, G., “Technical Gas Analvsis,” pp. 191-3, Nostrand, New York, iG34. (6) ”yerson~ A* L . ~Eicher~J. H . ~J . Am. Chem. SOC.74,2788-61 (1952). (7) PaPPW w.S.J Weber, c. pv., A N A L . CHEM.37,407-10 (1968). ( 8 ) Rodden, c. J.9 “Analytical Chemistry of the Manhattan Project,” 1st ed., p. 83, McGraw-Hill, New York, 1950.
(9) Weber, C. W.,ANAL.CHEM.32, 38791, 1960. (10) Woodall, He c., “Viscosity of Some Commercial Gases,” Engineering Data Sheet, Carbide and Carbon Chemical Company, K-25 1 7 J lg51* RECEIVEDfor review March 22, 1965. Accepted July 1, 1965. Division of Analytical Chemistry, 134th Meeting, ACS, Chicago, Ill., September, 1958. Work performed a t the Oak Ridge Gaseous Diffusion Plant operated by Union Carbide Corp. for the U. S. Atomic Energy Commission.
Liquid Scintillation Counting of Carbon-14 in Aqueous Digests of Whole Tissues RUSSELL TYE and J. DAVID ENGEL Kettering laboratory, Department o f Environmental Health, College o f Medicine, University o f Cincinnati, Cincinnati, Ohio
b The accuracy and precision of a method for the determination of carbon-14 in animal tissues and excreta has been evaluated. This method has the merit of being very general in applicability to different tissues and to different p emitters and is relatively simple in execution. Animal tissues may b e digested to near homogeneity in aqueous sodium hydroxide. Such digests are suitable for the determination of carbon-1 4 by liquid scintillation counting. Dispersions of the digests are stabilized by suspension upon Cab-0-Sil, in a scintillation medium consisting of dioxane, naphthalene, toluene, the scintillators 2,5-diphenyloxazole and 1,4-bis-2-(5-phenyloxazolyl)-benzene, and a compound to prevent freezing. In this medium urine may b e dissolved and its radioactivity counted without a gelling agent, but larger amounts may b e handled as a second phase, suspended upon Cab0-Sil. Carbon-14 in feces may b e determined by partial digestion in aqueous hydrochloric acid, mechanical homogenization, neutralization, and counting in suspension. Labeled cornpounds of widely differing polarities and characteristics of solubility have been used to show that the counting efficiency of the system is adequately independent of phase distribution or other phenomena affected by such differences.
I
THE COCRSE of studying the physiologic absorption and disposition of radiolabelled materials, it was desirable to obtain accurate analyses of whole tissues. A means of accomplishing this is to digest the tissues and to measure the radioactivity of dispersions of the digests by liquid scintillation counting. Thixotropic gels-e.g., Cab-
N
0-Sil-are convenient media for the dispersion of radioactive mixtures that are insufficiently soluble in the organic solvents used to dissolve available scintillators (3). However, uncertain distribution of radioactivity in the phases necessitates a counting system which is substantially independent of phase distribution. The problem of the influence of the distribution of unidentified compounds has been examined by comparison of the counting of cai bon-14 in three compounds of widely differing polarities. Acetic acid niay be expected t o be found distributed in significant proportions in both phases of a n organic aqueous system, and to be entirely within the aqueous phase (as the salt) a t high pH. The second compound, 4,4’ - methylenebis(2,6 - di - tert - butylphenol), abbreviated X B P , is highly insoluble in water a t any pH, but moderately soluble in organic liquids, whereas Banvel D, 2-1nethoxy-3~6-dichlorobenzoic acid, a coiiiniercial weed killer, has intermediate characteristics of solubility. EXPERIMENTAL
A11 counting was done by a Packard Tri-Carb automatic liquid scintillation spectrometer, Model 314X, with optimum amplifier voltages and a “window” setting of 10 t o 50 volts. T h e samples reported in t h e first three tables were counted in glass vials (low potassium, Packard Instrument Co.) , and t h e remainder in polyethylene vials. Reagents. Cab-0-Si1 was supplied by Packard Instrument Co. T h e scintillation medium ( I ) , (DTS), was prepared as follows: dioxane, 1 liter; toluene, 50 ml.; naphthalene, 100 grams; 2,5-diphenyloxazole (PPO) , “Scintillation grade,” Packard Instrument Co., 16 grams; 1,4-bis-2-(5Apparatus.
phenyloxazolyl) - benzene (POPOP), “Scintillation grade,” Packard Instrument Co., 1 gram. This mixture may be expected to freeze near 0” C. and in that region an antifreeze must be present. Ordinarily, the water from the sample functions as the necessary antifreeze. However, if the sample as prepared for counting contains less than 5% of water, water or alcohol should be added to make up the difference. Procedure. Tissues were prepared for counting by chopping weighed specimens with surgical scissors and warming with 0 . 5 N aqueous XaOH (4 inl. per gram of tissue, with 4 ml. minimum) with occasional swirling, on a 60” C. water bath. After 10 t o 20 minutes a substantially homogeneous solution, or an emulsion of f a t t y and aqueous phases, was produced. Samples mere weighed before and after warming, and any lost water mas replaced. The absence of substantial loss of water was taken as evidence that no loss of volatile metabolites had occurred. One-tenth to 0.5 ml. of the solution (or homogeneous emulsion) was added to 10 to 20 ml. of D T N in a 25-ml. polyethylene counting vial and shaken. Cab-0-Sil, in quantities sufficient to encompass the volume of DTX (-0.5 gram per 10 ml.), was added, and the sample \vas shaken vigorously for 1 minute. The emission of the sample was counted for periods of 10 minutes, several times in 24 hours, to allow for stabilization; then 0.1 nil. (activity approximately 10,000 c.p.m.) of a standardized solution of the radioactive compound in D T N was added from the 0.1-ml. pipet, the sample was again shaken thoroughly, and the emission was recounted. The quantity of radiotracer originally in the sample was then calculated on a relative basis. Urine was neutralized with HC1 and specimens ranging up to 1 ml. of urine or 1 ml. of aqueous solution of urine per 10 ml. (see discussion) were added to D T N for counting. If a n aqueous VOL. 37,
NO. 10,
SEPTEMBER 1965
0
1225
