V O L U M E 21, NO. 9, S E P T E M B E R 1 9 4 9
1059
reference material, depending on the nature of the energy change taking place. Figure 1 shows the rate of heating for the gluten and gelatin samples and the type of curves obtained for each protein studied when the reading of the differential thermal galvanometer was plotted against the temperature of the block. All three proteins studied undergo exothermic effects during heating. The temperature difference between the protein and reference niaterial was greater for gluten than for either gelatin or egg albumin. However, the exothermic effects take place over a wider temperature range for the gelatin and albumin. The maximum temperature difference for albumin occurred a t 100' C., for gelatin a t about 110' C., and for gluten a t about 130' C. The two small inflections on the gelatin cuive just below and just above 150" C. are probably not significant and may be due to fluctuations in heating rate. I t was assumed that the thermal curves would not be reversible on cooling, inasmuch as 200' C. TEMPERATURE is well above the temperature required to deFigure l. Differential Thermal Curves of Vacuum-Dried Proteins nature the proteins. Therefore, no cooling Straight line shows rate of heating for gluten and gelatin curves were made. Considerably more work will be required before it will be possible to discuss the exact significance of these difshowed that an increased rate of heatiiig not only increased the ferences. However, the results thus far obtained are reported magnitude of the dips and peaks but also caused the breaks to because the method promises to be useful in the study of the heat occur a t higher temperatures. There mas a time-temperature denaturation of proteins and the influence of moisture content interaction-Le., gluten protein denatured a t a lower temperature on the temperature a t which denaturation occurs. if heated over a longer period of time. The protein samples-ashless gelatin, powdered egg albumin, LITERATURE C I T E D and wheat gluten-were dried in a vacuum oven a t 40" C. for 48 hours before being analyzed. The preliminary drying was (1) Grim, R. E., and Rowland, R. A., J . Am. Ceramic Soc., 27, 05-76 necessary to prevent the endothermic effects accompanying loss (1944). (2) Le Chatelier, H.. Bull. SOC. franc. mine'ral., 10, 204-11 (1887); of absorbed water from overshadowing all other effects. The cited by Grim ( 1 ) . differential thermocouple, connected to a sensitive galvanom(3) Norton, F. H., J . Am. Ceramic Soc., 22, 54-6 (1939). eter (0.025' per scale), measures differences in the rate of (4) Orcel and CaillBre, Compt. rend., 147, 774-6 (1933). heating in the sample and reference material. Because both (5) Vold, 41.J., ANLL.CHEM.,21, 683 (1949). materials are in the same small copper block they will heat at RECEIVED December 8, 1918. This paper represents a portion of the work the same rate unless energy changes occur in the sample, in carried out under a cooperative research program by Pillsbury Mills, Ino., which case the sample will heat either faster or slower than the and the Ohio State University Research Foundation.
Determination of Radiophosphorus in Plant Material by Solution Counting CL.IYTON JIClULIFFE, Cornell L'niversity, Zthaca, S. Y .
I
T W A Gdesirable t u determine radiophosphorus in plant nia-
terial by some means other than counting from a uniformly deposited solid, in order to simplify and speed the analysis without loss of accuracy. MacKenzie and Dean (9) dcveloped an accurate method of analysis for PB1and Pa2 by precipitating phosphorus as ammonium phosphomolybdatc and then reprecipitating as magnesium ammonium phosphate; the latter precipitate was collected as 2 thin uniform layer on a filter ring under carefully standardized conditions. This procedure gives good results, hut it is tinie-consuming and the uniform layer for counting must be carefully prepared. If the layer becomes too thick, it is necessary to make self-absorption corrections. The area of the deposit must be precisely reproduced and geometry with respect to the Geiger.1Iuller counter closely maintained. The method for determing P32in solutions presented here is
much more rapid, overcomes most of the disadvantages just mentioned, andisaccurate toastandard deviation of 0.770. Other liquid counters and procedures for counting from solution are described by Olson et al. ( I O ) , Barnes ( 2 ) )Bale et al. ( I ) , Wang et al. ( l 4 ) , Barnes and Salley (5),Comer and Seller ( 5 ) , and Veal1 ( I S ) . PREPARATION O F SOLUTION FOR COUNTING
Weighed samples of plant material containing from 1 to 30 mg. of phosphorus were heated in 50-ml. Pyrex beakers in an electric muffle a t 300" C. for a t least 6 hours to destroy organic matter. Nitric acid (8 S ) was added and evaporated to dryness and the residue was heated a t 400' C. for 15 minutes to complete the destruction of organic matter. Silica was dehydrated with concentrated hydrochloric acid. The residue was taken up in 2.5 ml. of 2 K nitric acid and transferred tvith hot water to a 23-ml. volumetric flask if the specific activity of the phosphorus was
ANALYTICAL CHEMISTRY A rapid method for the determination of Pdzin solution is presented, accurate to a standard deviation of a single measure of less than 1%. The Geiger-MCller counter assembly is described, and the preparation of the sample is given along with the counting procedure. The preparation of uranium standards is presented and their use as efficiency standards and for the determination of the resolving time of the counter is given. Factors influencing the count, such as solution volume, K 4 O background, temperature, self-absorption, and adsorption of isotopes on glass, are discussed. The efFirienry o f the liquid counter is rompared with the thin end-window counter.
high. When the specific activity was low, 10 ml. of 2 N nitric acid were accurately pipetted into the 50-ml. beaker and carefully stirred and the suspension was transferred without washing to a 15-ml. centrifuge tube. The silica was centrifuged down or, in the case of the volumetric flasks, allowed to settle for 24 hours. These solutions were then counted. If the count exceeded 10,000 counts per minute, the solutions were diluted. Total phosphorus was determined by the molybdivanadophosphoric acid method (8) on an aliquot of the solution used for counting. When the specific activity was high, the colored solution on which the total phosphorus was determined was used for rnunting.
GEIGER-MLYLLER
.
COUNTER ASSEMBLY
Figure 1 is a cross-sectional view of the Geiger-Muller countei and its surrounding cup. The unfilled counter is the same as that used by Solomon and Estes (12). The shell was filled with 1.5 cm. of absolute ethyl alcohol and 8.5 cm. of argon, giving a self-quenched counter with a plateau 200 volts in width and a slope of 3.5% per 100 volts, The counter was slightly light-sensitive and was covered with a black cloth during the count. The background was 25 counts per minute when the cup was filled with distilled water. Shielding with 5 em. (2 inches) of lead should reduce the background count by at least a factor of two. The Geiger-Miiller counter was clamped tightly to a rigid ring stand a t the top of the tube just below where the tungsten leads emerged and a t the top of the filling tube. Rubber served as a cushion between the clamps and the glass, yet held the counter tightly. The cup was centered with the counter and clamped tightly in position. Thus, the geometry of the cup with reference to the Geiger-Muller counter was fixed and it was not necessary to have an elaborate centering mechanism. A pinch clamp on the rubber tubing closed the cup a t the bottom. A small funnel was mounted on a swivel directly below the cup. Washings and solutions which were not saved were drained into this funnel, and the solution was conducted by means of rubber tubing into a 20-liter bottle on the floor. The funnel was swung from under the cup when the solution waF to be saved. A 20-liter bottle of distilled water was mounted above and behind the counter, the water being used to rinse the cup. With the spacing between the counter and cup bet,ween 1.5 and 2.0 mm. the cup held approximately 9 ml. when filled 1.5 cm. above the top of the silvered portion of the counter.
ards did show adsorption on the glass surface. After rinsing the cup thoroughly with water, 200 to 250 counts per minute ovei the background remained from a uranium nitrate solution giving 12,000 counts per minute. Sodium or aininonium tartrate added in equal molar quantities to the uranium nitrate solution formed a soluble complex which coniplrtrly prevented the adsorption of uranium. EFFICIENCY STANDARDS AND RESOLVING TIME MEASUREMENT
Eranium nitrate standards were prepared by hesting pure uranyl acetate to constant weight at 700" C. to form UsO,, a good primary standard. The oxide was converted to the nitrate by the addition of concentrated nitric acid, after covering the oxide witb water to slow the reaction, and subsequent evaporation to dryness. Two standards were prepared, one containing 24 grams of Us08 per liter, the second 12 grams. Twenty and 35 grams of sodium tartrate were added to complex the uranium and prevent. ite adsorption on glass surfaces. The larger quantity of sodium tartrate was added to the smaller uranium standard in order to give approximately the same solution density and self-absorption of the beta-particles. Thus, the difference in the self-absorption between the two uranium standards is eliminated and need not be considered in the ca1cul:ition of the resolving time. These two standards were counted a t regular int>ervalsto determine any changes that might have occurred in the Geiger-Muller counter, scaling circuit, or register-i.e., efficiency standards to determine if the same count was obtained from day to day. One standard counted approximately 11,000 per minute under the conditions described in this paper. The second counted a little over one half this value. With these uranium standards, the resolving time can be determined easily by a modification of the combined source method
(4,1 1 ) : FLEXIBLE BRAIDED COPPER LEAD5 THICK TUNGSTEN LEADS
FILLING TUBE
PROCEDURE FOR COUNTING
Prior to counting a series of samples, the cup was filled with chromic acid cleaning solution and rinsed to assure good drainage. The cup was rinsed with the sample to be counted by filling the cup and draining. If the samples being counted have approximately the same specific act,ivity, it is not necessary to rinse the cup with the next sample. Draining the cup and then taking a count without rinsing revealed that less t,han 1% of the solut,ion remained. Thus, if two samples have counting rates differing as much as 5070, the error introduced nil1 be less than 0.5%. Rinsing by filling and draining the cup four times with water removed all traces of 50,000 counts per minute from P 3 2 ; this indicated there was no adsorption of phosphorus in acid solutions (0.01 S nitric acid or stronger) on the glass walls of the counter or cup when working with specific activities of the order found in plant mat,erial or fertilizers. When working with extremely high specific activities such a s carrier-free phosphorus, there may be adsorption on glass surfaces ( 7 ) , but this might be prevented by complexing the phosphorus. Uranium nitrate or acetate s~lutinnrused as permanent ;rand-
FUNNEL
I
y
(0.005''
TUNGSTEN
1.5-2.0 mm.
SILVER PLATE -GLASS
RUBBER TUBING
Figure 1.
7
CUP
DIAMETER OF COUNTER = 19rnrn.
Geiger-JICller Counter and Cup
1061
V O L U M E 21, NO. 9, S E P T E M B E R 1 9 4 9
+
- (C, B ) C,(C, +B)
2CI rd
=
where T , = resolving time, C1 = observed counting rate of loa liranium standard, C2 = observed counting rate of high uranium Itandard, and B = background count. The background count is added t o Czto compensate for the second background count which appears in the 2C1term. Using this resolving time, the true counting rate is then given by (4): ~
here C = observed counting rate, CC = true counting rate, and = rrsolving time. To maintain good precision it is n-ise not to exceed 10,OOO counts per minute, because the error in the resolving time correction may become significant above this rate. ii
Z’,
ADDITIONAL F A W R S INFLUENCING THE COUNT
Solution Volume. The cup is filled with the sample to a height least 0.5 cm. above the silvered portion of the counter. (‘ountp taken at 0.5 and 2 cm. above the silvered portion were the same. Therefore, precautions need only be taken to assure that the silvered area is covered. When working with isotope% mom which most of the count is from gamma-rays, this may not he true. Increased Background from K40. One of the isotopes of potassium, KM, is naturally radioactive and its presence in a solution adds to the count of the sample. Actually potassium hae been determined by its radiation (3, 6 ) . This K@activity, how. ever, is low and may be disregarded unless the P32sample count is tmly a few times background. If necessary, K40 may be deterriiiried by counting a plant sample which has not received P32 and iisrd as the counter background, Or, if the ignition temperature ,>f the plant material is increased several hundred degrees, most of the potassium should be lost without losing phosphorus. Temperature. Variations in temperature have an effect on r tit. characteristics of the Geiger counter, making it necessary to rnaintain solution temperatures fairly constant. A change from 24” to 28” C. gave an error of less than 1%. The surface Area of the solution in the cup is small, and with water a t room temperature the error introduced by evaporation is negligible as memured by counting repeatedly a standard over a 2-hour inter-
upported i n part by a grant from the Industr~ t’hmphate Research Committee. i‘Sauchelli, . chairman. LITERATURE CITED
Bale, R. F.,Haven, F. L., and LrFevre. M. L., Rm.Sci. Instruments, 10,193 (1939).
Barnes, A. H., Ibid., 7, 106 (1938). Barnes, R. B., and Salley, D. J.. IND.EICG.CHEM.,ANAL.F h . 15, 4 (1943)
Beers, Y . , Rev.Sci. Instruments, 13, 73 (1942). Comer, C . L., and Neller, J. R.. Plant Physiol., 22, 174 (1947). Gaudin, A . AI., and Pannell. .J. H , , ANAL. CHEM.,20, 1154 (1945).
Hall, N. S.,and MacKenzie. A . J . , Soil Sei. Soc. A m . , Proc., 12 101 (1947).
Kitson, R. E., and Mellori, M.G . , IKD.ENG.CHEM.,ANAL.ED.. 16, 379 (1944).
MacKenzie, A . J., and Dean, L. A , , ANAL.CHEM., 20,559 (1948, Olson, A. R., Libby, W. F., Long, F. A , , and Halford, R. S., J Am. Chem. SOC.,58, 1313 (1936).
Reid, -4.F., in Wilson, D. W., Nier, A. 0. C., and Reimann, S.P.. “Preparation and Measurement of Isotopic Tracers,” p. 83. Ann Arbor, Mich., J. W.Edwards, 1946. Solomon, -4.K., and Estes, H. D., Reo. Sci. Instrtments, 19. 47 (1948).
Veall, K.,Brit. J . Radiology, 21, 347 (1948). Wang, J. C., Marvin, J. F., and Stenstrom, K. W.,Reu S m Instruments, 13, 81 (1942). RECEIVED
January 28. 1949
