Procedure for Routine Assay of Tritium in Water C. GARDNER SWAIN, V. P. KREITER, and WILLIAM A. SHEPPARD Massachusetts lnstitute o f Technology, Cambridge, Mass.
A precise method for the assay of the tritium content of tritiated water has been developed. The method involves reduction of ca. 1 millimole of the tritiated water to tritiated hydrogen which is transferred to an ionization chamber. The ion current activity of the tritium is measured with a vibrating reed electrometer. Analyses are reproducible to &2% over a thousandto lo-$ curies permillimole. fold activity range, from T
VIE\\- of the increasing-~ iniDortance of tritium in chemical 1.and biological studies 7 ) a precise method is reported for routine assay of tritium in water samples. The method inS
(6,
the
volves reduction of approximately 1 millimole of water to hydrogenwith amalgamated magnesium a t 400" C. in a borosilicate glass bomb. The hydrogen produced is transferred to an ionization chamber in which the tritium activity is determined with a vibrating reed electrometer. Because excellent papers have appeared recently on reduction of organic compounds to gas for tritium assay ( 8 ) and on ion current measurement ( 1 , 9) this procedure is limited to reducing water and to transferring the gas to an ionization chamber. This is a simplification of the procedure described by Henriques and Margnetti ( 4 ) . EXPERIMENTAL
structed with a 19/38 standard-taper ground-glass joint, a. Handle B, designed t o fit into a side arm on the bomb holder and constructed with a ring tip, was made from an inner 10/30 standard-taper ground-glass joint with sealed tube. Toepler pump, T , consists of bulbs that may be filled with mercury from a reservoir so that the volume of the line may be varied. The total volume of the line-up to the stopcocks, F and G , and calibration mark b, including manometer M , is approximately 60 ml., while the volumes between b and c, c and d, and d and e are 50, 50, and 100 ml., respectively. These volumes were determined precise1.y by application of the gas law. A measured volume of dry air a t a known pressure was introduced into the evacuated syFtem from gas buret, K , and the pressure in the system was measured on manometer M. This manometer is 1 meter high and the quantitv of mercury in it was adjusted for each reading, so that the right arm of the manometer was always set a t the same point. The total volume between stopcocks G and H , and in the ionization chamber, C (approximately 250 ml.), was also determined precisely by the calibration procedure. The bomb, after cooling and weighing, was placed in bomb holder A , so that the tip of the bomb fitted into the hole in the ring tip of handle B. The remainder of the bomb was surrounded with copper gauze to prevent shattering of the bomb holder when the bomb was broken. The system including the ionization chamber was evacuated to a pressure of a t least 10-2 mm. of mercury, and all stopcocks were then closed. By txisting handle B , the bomb tip was broken. Stopcocks D and E were opened and with the mercury level in Toepler pump T adjusted to calibration mark b, the ressure a a s determined on the manometer. The number ofmoles of hydrogen gas was calculated from the gas law using the room temperature, the measured pressure and known volume (corrected for the volume displaced by the bomb n hich was calculated from the weight of the bomb and the density of 2.24 grams per cc. for glass). As a check, the mercury level in Toepler pump 5" was adjusted a t other calibration points (c, d, or e ) , so that the amount of hydrogen could be calculated using pressure readings a t other calibrated volumw. The tritiated hydrogen was now expanded into the ionization chamber, C, by opening stopcocks G and H . In order to eliminate the fractionation of tritium by diffusion in the line, the mercury in Toepler pump T was moved up and down several times between levels b and e, and the gas subsequently allowed to stand for 15 minutes to permit equilibration of the tritium and hydrogen before commencing the next operation. The fraction of the tritiated hydrogen sample introduced into the chamber can be regulated somewhat by adjustment of the mercury level in the Toepler pump, T,to different calibration level?. The ionization chamber, C, was filled to atmospheric presfiure with tank hydrogen by use of mercury valve V . The details of construction and operation of this valve are described by Neville (6). The valve from the hydrogen tank was opened (the rate
Equipment and Materials- Aliquots of a stock solution of tritiated water of activity i pc. per ml. were quantitatively diluted by !wight in order to prepare a series of water solutions containing a range of tritium activities. The magnesium was Baker and Adamson C.P. granular grade. The tank hydrogen was Airco regular grade of 99.5% purity. Aivacuum line of standard design, employing a one-stage oil diffusion pump backed by a Welch Duoseal oil pump provided a vacuum of 10-4 to 10-6 mm. of mercury. The line designed for the transfer of tritiated hydrogen to the ionization chamber is shown in Figure 1. The Borkowski type ionization chambers ( 1 ) and the vibrating reed electrometer (RIodel30) coupled to a Brown potentiometric strip-chart recorder with multiple range recording (Model 39) are of standard design available from Applied Physics Corp., Pasadena, Calif., for carbon-14 analysis (6). All equi ment was in an air-conditioned room maintained a t 26" A 1 C. and < 45% humidity. Reduction of Tritiated Water. A sample of tritiated water (13 to 18 mn.. 0.7 to 1.0 mmole) was sealed in a small weighed borosilicate glass ampoule constructed with a break tip (Figure 2). The filling was accomplished by placing the open tip of a heated ampoule in the tritiated water and allowing a sample of the water to be drawn into the ampoule as it was allowed to cool. The tip was sealed in a small flame. The weight of the sample was determined using an ordinary analytical balance and the ampoule was placed in the bomb shovn in Figure 2, with 0.3 gram of granular magnesium and 0.3 gram of mercury. The bomb was constructed of 10-mm. tubing (ordinary Pyrex, Corning KO.774) with one end sealed with a break tip. The bomb was connected to the auxiliary vacuum line with pressure tubing, evacuated to a pressure of 0.05 mm. of mercury, sealed a t the constriction, and shaken to mix the mercury and the magnesium thoroughly and to break the ampoule containing the tritiated water. The bomb was then placed in a muffle furnace (Blue c M electric furnace, hfodel M225A) regulated a t VACUUM a temperature of 400" to 410' C. for 1 to 2 hours. Transfer of Tritiated Hydrogen to IonizaFigure 1. Vacuum line for transfer of tritiated hydrogen to tion Chamber. The vacuum line is shown in Figure 1. The bomb holder, A , is conionization chamber
B
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1157
1158
ANALYTICAL CHEMISTRY
of flow of hydrogen had been previously adjusted so that C filled t o atmospheric pressure in 10 to 15 seconds) and stopcock G was turned so that the tank hydrogen was allowed to flow into C through the medium-porosity fritted-glass disk of valve V . As soon as the pressure in C reached atmospheric, as indicated by cessation in the movement of the mercury in valve V and by the recommencement of bubbling of hydrogen through safety bubbler P, stopcock H was closed, and the chamber, C, was removed. Although counting characteristics vary considerably with the internal pressure with certain gases in the chamber ( I ) , this effect is reported as unimportant for hydrogen in the region of atmospheric pressure (9). Tritium A s s a y . T h e i o n i z a t i o n chamber was connected to the vibrating reed electrometer and the activity determined in the standard manner (1, 9 ) by measurement of the rate of charge of a condenser or of the voltage drop across a standard high precision resistance, using 180-volt ion collecting potential across the chamber. To calculate the specific activity, the measured activity was divided by the millimoles of tritiated hydrogen. The tritiated hydrogen was calculated from the sample weight (in millimoles of hvdrogen) times the volume fraction of the ionization chamber compared to the volume of the whole system. Volumetric measurement of the millimoles of hydrogen gas is employed only as a check on the yield in reduction and consequently measurement of the gas pressure is not necessary for routine analysis. On completion of the assay, the tritiated hydrogen gas was evacuated from the chamber and the chamber was flushed a t least three times by filling with dry air and re-evacuating. The vacuum line was also flushed in a similar manner, employing an auxiliary vacuum system for exhausting the tritiated hydrogen. All tritiated hydrogen gas was exhausted into a ventilating system and the bomb with a Fig re small amount of adsorbed tritium was Construction discarded (8). No contamination of the of bomb vacuum line or the ionization chambers with tritium ivas detected.
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RESULTS
A study of the effect of time of heating on the yield of gas and on the specific counting rate was made (Table I). The yield of gas appeared essentially quantitative after 5 minutes st 400" C., and was almost invariably greater than 100% after 1 t o 2 hours of heating. A considerable amount of gas (of the order of 5 t o 10% of the total amount obtained from 18-mg. water samples) was found to be evolved when only magnesium amalgam was heated in an evacuated bomb. Although this suggests that the reduction of water samples is not over 90 to 95% complete, the effect on reproducibility seems negligible] since the specific counting rate obtained after 1 to 2 hours of heating is reproducible to 1 to 2% (Tables I and 11). That some tritium is retained in the
Table I.
Effect of Time of Heating at 400" C. on Yield of Gas and Specific Counting Rate
Water Sample Gaa Size, Yielda, Mg. 7% 56 21.1 47.2 51 4 16.7 !b 14.3 106 6 5 104 1 15.2 15 15 0 106 2 16 15.8 102 7 15 16 8 101 3 15 16 8 104 6 16 7 98 4 25 45 21.6 96 2 102 8 14 8 47 60 13.9 101 5 120 14 1 103 7 120 14 5 100 5 87 9 1020 15.1 1200 21.0 88 5 a Based on weight of water sample. 6 At 180" C. Heating Time, Min.
Specific Counting Rate, Mv. per Mmole Based on Based on mmoles of wt. of gaa water 263 125 263 135 462 493 492 513 487 517 499 512 509 502 510 487 506 498 514 535 533 514 511 520 530 512 515 519 528 463 512 453
reduction mixture even after 2 hours of heating was determined from the fact that reduction of samples of the same tritiated water using zinc and nickelic oxide a t 640' C. (8) yielded gas of a specific counting rate about 12% higher. Although the reduction appeared to have reached its maximum value after onIy a few minutes, the highest yield of tritium in the gas was obtained only after approximately I-hour heating. The reduction mixture should not be heated beyond 2 to 3 hours, since loss of gas occurs (6).
The usefulness of the tritium assay procedure was tested by analyzing a series of water samples having an activity variation of a thousandfold. The mean specific counting rate and average deviation from the mean for each solution are reported in Table 11. The method employing voltage drop across a standard resistance was more precise a t high activity levels. In general, a precision of better than i.2% was obtained for solutions of activity of over pc. per mmole. A slightly lower precision was obtained a t the pc. per mmole level. The specific counting rate was found to be a linear function of the tritium concentration (9) with a precision of the order of & l to 2%. In general, the reproducibility of the specific counting rates expressed in terms of millimoles of water was better than that expressed in terms of millimoles of gas. This is reasonable in view of the fact that the large. amounts of gas evolved from the magnesium should make the number of millimoles of gas more variable than would be expected from an error in weighing the water. In routine analysis, measurements of the millimoles of gas should not be necessary and the results may be reported solely in terms of millimoles of water. In any given tracer experiment the highest possible precision in measuring the specific counting rates by this procedure will
Table 11. Tritium Assay Results" Tritium Actlvlty in Water, IIc,per Mmole 1.39 0.276 0,0691 0.0155 0.00144
Mv.per ZImoleb
Gad Wa tere 518 j, 6 1 524 1. 9 101 2 103 I 1 26 5 h 0 . 4 2 6 . 3 ?= 0 . 4
+
I Q
hleans
I I
M v . per pc. Gasd Water' 372 1. 4 377f 6 368 + 5 375 f 2 38Ag+ 6 381 f 5 I
I
I
375 i 6
378 f 2
Specific Counting Rates M v . per Sec. per Mmolec Gasd Watere 29.3 f0 . 4 29.8 I 0 . 5 5 61 f 0 . 0 9 5.72 1 . 0 . 0 2 1.49 ZtO.04 1 50 f 0 . 0 2 0 . 3 1 7 3 0.002 ~ 0 327f 0.001 0.0303 f 0 . 0 0 0 8 0.0314 + 0.0008
MY. per Sec. per pc. Gasd Watere 21.1 h 0 . 3 2 1 . 4 i. 0 . 3 20.4 0 . 3 20.7 f 0 . 1 21.6 + 0.3 21.7 0.3 20 4 =t0 . 1 21.1 + 0 . 1 21.0 + 0.6 21.8 0.5 20 9 ?= 0 . 4 2 1 . 3 Zt 0 . 4
*
+
No. of Samples 8 5
4
3 4
Data obtained by heating water samples of 16- to 20-mg. weight at approximately 400' C. for times varying from 2 t o 3 hours. Two ion chambers were used interchangeably. Data were obtained by two different workers (V. P. K . and W. A. S.). b From voltage drop acrose standard l O l ~ - o h mresistance. c From rate of charge of a condenser. d Based on mmoles of gas. 0 Based on mmoles of water sample. I Precision expressed &eaverage deviation from mean. I Ion current too low to use voltage drop method. 0
1159
V O L U M E 2 7 , N O , 7, J U L Y 1 9 5 5 probably be obtained by carrying out the reductions for 1 to 2 hours a t 400’ C. I n addition, the size of the water samples should be in the range of 13 to 18 mg. and should not be over 20 mg. I n a few cases in which large samples of water were used (in the order of 25 to 30 mg.), the yield of gas was only 90 to 95% and the specific counting rate was inconsistent with that obtained from smaller samples. The lowest level of tritium activity a t which measurements were made was 10-3 pc, per mmole of water. At this level the ion current produced by the tritium is approximately ten times that produced by the background. With greater care and expc. per mmole tended counting periods, activities as low as can be measured, although a t such a level the precision is expected to be relatively poor because the ion current produced by the tritium is of the same order as the background. The precision a t this low level can probably be improved by modifying the apparatus, so that the hydrogen from larger samples of tritiated water can be introduced into the ion chamber ( 4 ) . Tritiated water samples of much higher specific activity than those used in this work also may be analyzed in this apparatus (9). The complete working time for an analysis including weighing the water sample is approximately 45 minutes. The method has the advantage that the only important contamination is in the bomb tube (8) which is discarded, so that special procedures such ae preconditioning of apparatus (3) are avoided. Another advantage is that the sealed tubes are not constructed of special boro-
silicate glass and do not require the higher temperature heating (8). Reduction with zinc a t 400” C. ( 2 ) should also be practical for tritium assay, but it was not examined in this investigation. ACKNOWLEDGMENT
The authors are indebted t o I. 4. Berstein of Tracerlab, Inc., Boston, for helpful discussion a t the outset of this work. LITERATURE CITED (1)
Brownell, G. L., and Lockhart, H. S.,Nucleonics, 10, No. 2 , 26 (1952).
(2) Chinard, F. P., and Enns, T., ASAL. CHEM., 25, 1413 (1953).
Fukushima, D. K., Kritchevsky, T. H., Eidinoff, 11.L., and GaIlagher, T. F., J . Am. Chem. Soc., 7 4 , 4 8 7 (1952). (4) Henriques, F. C., Jr., and Nargnetti, C., ISD. ENG.CHEY., (3)
A N ~ LED., . 18, 420 (1946).
Kamen, 31. D., “Radioactive Tracers in Biology,” 2nd ed.., Chap. VII, Academic, New York, 1951. (6) Neville, 0. K., J. Am. Chem. Soc., 70, 3499 (1948). (7) Thomas, S.L., and Turner, H. S., Quart. Reus. ( L o n d o n ) , 7 , 411 (5)
(1953). (8)
Wilzbach, K. E., Kaplan, L., and Brown, W. G., Science, 118,
(9)
Wilzbach, K. E., Van Dyken, A. R., and Kaplan, L., AXAL. CHEM.,26, 880 (1954).
522 (1953).
RECEIVED for review June 1 , 1954. Accepted December 29, 1954. This work was supported by the research program of the Atomic Energy Commission.
Determination of Tannins and Related Polyphenols in Foods Comparison of Loewenthal and Pro Methods CHRISTIAN J. B. SMIT, MAYNARD A. JOSLYN, and AARON LUKTON Food Technology Department, University of California, Berkeley, Calif.
The volumetric permanganate titration and the colorimetric phosphomolybdic-tungstate reduction procedure were compared for pure polyphenols, commercial tannins, and partially purified fruit tannins. The permanganate titration gave significantly higher results for catechol, hydroquinone, pyrogallol, and chlorogenic acid, and lower results for phenol, resorcinol, catechin, and quercetin. With commercial tannin preparations the results were lower but were essentially similar for fruit tannins. Changes in absorption spectra during titration with permanganate and changes in redox potential are reported.
T
HE available methods of audvsis for tannins have been critically reviened by Joslyn ( 5 ) , Mitchell (?), and Sieren-
stein ( 9 ) . For the determination of tannins and related polyphenols present in fruits and fruit products, the t n o most widely used are the Loewenthal volumetric procedure ( 1) and the FolinDenis colorimetric procedure (10, 12). I n the present investigation these two general methods of determination of tannins were compared for a series of known compounds as well as a group of commercial tannins and isolated fruit tannins. The volumetric permanganate method developed by Loewenthal ( 6 ) , after some modification, was adopted as an official method for tannins in coffee and tea, spices and condiments, and wines. Loewenthal improved the method originally proposed by Rlonier (a), in which the tannin was titrated directly with potassium permanganate, by carrying out the titration in the presence of indigo carmine. Loewenthal stated that the indigo carmine acted not as an indicator, but as a regulator, because in
its presence only those compounds were oxidized whose rates of reaction with permanganate were faster than with indigo carmine. T o determine the nontannin equivalent, a gelatin solution with salt was used to precipitate the tannins. Proctor (11 ) repeated Loewenthal’s work and made improvements in standardizat,ion of the permanganate solution. I n the determination of tannin in coffee and tea, gelatin is used to separate tannin from solution and other permanganate reducing substances present’. I n wines, purified bone black is used to absorb tannins and coloring matter. During the titration the permanganate is added in 1-ml. quantities with constant stirring until a green color is reached. The titration is then continued dropwise to a golden yellow end point. The inclusion of coloring mat,ter with tannin in the analysis of wine \vas supported by Bate-Smith and Swain’s (3)contention that leucoanthocyanins are closely related chemically to the catechins and should be regarded as prototypes of condensed tannins. Williams ( 1 4 ) showed by paper chromatography that leucoanthocyanins, catechins, and chlorogenic acid, present in cider, all contribute to the total tannin titration. Hide powder removed the major part of the simple polyphenols present and the hulk of the other more complex phenolic materials. Barua and Roberts (2) criticized the Loewenthal procedure because of the errors involved in the arbitrary end point. They found the method became completely unreliable with oxidized tannins, because the titration was carried to a different end point owing to decreases in the rate of oxidation. The Folin and Denis ( 4 ) colorimetric procedure as modified by Rosenblatt and Peluso (12) and Pro (10) is official for tannins in distilled liquors. The blue color which is produced only after addition of alkali to a mixture of the Folin-Denis reagent with
