V O L U M E 27, NO. 2, F E B R U A R Y 1 9 5 5 Table I.
Weights of Mercur? Deposited on and Evaporated from Samples
samDifference -0.3
295
ple
So.
sam- - Hg, AIg.
Hg, l f g . Deposited Evap.
Diiference 0 -0.7 -0.3
-0.1 -0.1 0 0 .I 0 0 0 2
0
0 0 -0 4 0 .5 0 .3
0 0 -0.1 0 -0.2 -0.4 -0.1 -0.5 0 0.1 0 -0.3 0 -0.3 0 0.2 0.2
-0 1 0
-0.8
-0.5 -0.4
-0.2 0 -0 1
O R
-0.2 0.1 0 -0 6
0
0.1 -0 6
such that the liquid level wa5 above the spherical portion of the flask. Every 5 or 10 minutes the flask was gently agitated. hfter being immersed in the oil bath for 30 t o 35 minutes, the flask was allowed to cool in air to room temperature while the vacuum was maintained. The samples were then individually reweighed to the nearest 0.1 nig., and the weight of mercury lost from each sample was thrn determined by difference. These data are presented in Table I. T h a t the weight loss suffered on heating Tyas due predominantly to the evapointion of mercury was indicated by the follorving experiment: Fifteen tinned-copper samples were numbered, v-eighed, and subjected to the above test procedure, including agitation. The maximum change in weight amounted t o 0.2 mg., the average being 0.1 1 mg. RESULTS 4\D DISCUSSIOY
T h e data in Table I indicate that the average difference betneen the weight of mercury depopited per piece (about 2 mg.) and the eight of mercury estimated by this method is & 0.21 mg. This amounts to an error of apprownately IO'%. The
ple So. 51 52 23
Deposited
5.1
sJ6 .5 7 58 39 bo
61 fi2 fi3
64 65 66
67 68 69 70 71 72 73 74 7.5
1.7 1.7 2.0 1.3 1 7 2.0 1.5 1 5 2.0 1.6 1.0
2.0 2 0 2.4 2.3 2 0 2 3 1.9 1.5
Evap. 2 0 2 3 2 0 2 0
1.7 2 2 2.4 "3 2 2 1.7 1 5 2.0 2 1 2.2 2.0 2.2 1.8 1.9 1.6
2.1 2.0 1.2
1.7 2.0
1.9
1.9 1 8 2.5
1.4 2.3
1.4
Difference
Sampie To.
-0.3 -0.6 0 -0.7 0 -0.2 -0 9 -0.8 -0.2 -0.1 -0.5 0
7ti
-0.1
88 89 $10 ')1 rc2 93
0.2 0.3 -0.2 0 .5 0 -0.1 0.4 0 -0.2 0 -0.4 -0.2
77
78 7:) 80 81 82 83 84 8.5 8A
HE, L l g Deposited 1.5 2.2 2.2 1.8 1.5 2.0 2.2
Evap. 1.7
2.2 2.2 2.3
1.5
97 98 99 100
2.0 1.4 2.3 2.2 1.9 1.7 2.3 1.7
2.1 2.5 1.7 1.6 2.0 1.7 2.1 2.0 2.0 2.6 2.6 0.8 1.9 2.2 2.3 2.2 1.9 1.7 2.3 1.8
Total
186.3
199.6
si
94 95 96
1.4
1.0 2.0 1.7 2.1 1 5 1.9 2.6 2.0 1. o
Difference -0.2 0 0 -0 5 0 -0 1 -0.3 -0 3 -0 6 0 0 0 -0 5 -0 1 0 -0 6 0.2 0 1
-0.8 0 0 0 0 0 -0.1
k20.9
probable error is f 0 19 mg. as computed by standard statistical procedures The total n eight of mercury added, as determined by the sum of the weights of mercury removed, was 0.1996 gram. This checks remarkably the actual 4 eight of mercury used for tumbling. However, the total weight of mercury added, as determined by the sum of the weights of mercury deposited, amounts to 0.1863 gram, indicating an appreciable R eighing error during preparation of samples. Since preparing the samples containing knon n weights of mercury necessitates an additional weighing compared with the test procedure for unknon n samples, the data reported here probably represent an abnormally high error. Larger quantities of mercury could no doubt be estimated more accurately. LITER4TURE CITED
(1) Eschka, A . , Dengler's Polytech. J., 204, 47 (1872). (2) Hallowvny, G . T.,Analyst, 31, 66 (1906). RECEIVED fcr review March 5 , 1954
Accepted September 29, 1954
Colorimetric Determination of Trace Quantities of Boric Acid in Biological Materials W. C. SMITH, JR., A. J. GOUDIE, Johnson
J. N. SIVERTSON Brunswick, N. J.
and
& Johnson Research Center, N e w
In a rapid and accurate method for the determination
of trace quantities of boric acid in small amounts of biological materials such as blood, urine, and animal tissue, organic matter is destroyed by fusion of the sample with lithium carbonate. The fusion mixture is dissolved in hydrochloric acid; then sulfuric acid is added, followed by a solution of carminic acid. The color develops within 5 minutes. Inorganic materials that are normally found in blood, urine, and animal tissue do not appear to interfere appreciably. Using the Beckman DZi spectrophotometer, the method is capable of determining from 2 to 15 y of boron with an accuracy within &I .Or.
I
X' T H E course of evaluating physiological effects of Iioric acid, it was necessary to determine the boric acid content in small samples of biological material (blood, urine, and animal tissue) in concentrations ranging upward from 1.0 p.p,m, (calculated as boron). Previous workers have developed a varic,ty of methods by which boron has been determined in soils, metals, and various organic materials. Most of these procedures n ere based on one of the following techniques: identical pH titrations, fluorescence measurements, spectrographic methods, and co!orimetric procedure#. When the identical p H electrometric procedure of m7ilc0~(16) was attempted in this laboratory, erratic results were obtained as reported by Berger and Truog (2), since the total quantity of boron available for each analysis wa8 usually less than 10 y .
ANALYTICAL CHEMISTRY
296
It was necessary to work with such small samples ( 2 grams or less) because only limited quantities of material were available from the physiological study referred to above. White ( 1 4 ) and his coworkers recommended determining boric acid by allowing it to react with benzoin and measuring the fluorescence produced by irradiating the complex with ultraviolet light. -4lthough this method is capable of yielding good results under ideal conditions, its accuracy is affected adversely by many of the inorganic ions normally found in biological materials. ai4
I
i
I
I
i
I
1
ai 2
ai0
0.08
u,06
I
9
w"PO4
EFFEOT OF TIME ON
-
COLOR OEVELOFMENT
AND
INTLNS I T Y Carmlnlc a d d r l t h l 8 p a d boron boron
ce C a r r n m R.d N.F wlthS,0,ag,ol
0,o 2
C
IO
20
so
1
40 50 TIME IN MINUTES
60
70
BO
Figure 1
Furthermore, a t the low concentrations of boron present in the biological materials, i t would be necessary to use fairly large samples so that the amounts of boron available for analysis would satisfy the sensitivity requirements of the method. T o use this method, therefore, i t a.ould be necessary to earn' out lengthy separations and concentration procedures and to use larger samples than are generally convenient with biological materials; hence, the search was continued for a more rapid and sensitive method. An attempt was made t o determine the boron spectrographically, as recommended by Smith, Schrenk, and King ( 9 ) )hIelvin and O'Connor ( 8 ) , and Steadman (10). This technique was extensively investigated, but. as reported by Melvin and O'Connor, the results were erratic. For example, in a typical series of 15 analyses where the boron concentration in human blood was varied between 1.0 and 10.8 p.p.m., the following relationship was found between the concentration of boron added and recovered (regression line of concentration recovered, R, on concentration added, A ) : R = 1.42.4 - 0.80. The standard error of estimate ( 1 6 ) of the concentration recovered is 4.01 p.p.m. Therefore, to include about 95% of the points, twice the qtandard error of estimate must be used. Thus, R could be expccted to fall within the limits 3 ~ 8 . 0 2p.p.m. 19 times out of 20. A s this error was so large, it n a s felt that too much additional work would be required to improve the precision of the spectrographic method. More promising results vxre obtained by investigating the numerous colorimetric methods reported in the Iiteraturc (1, 3-6, 11). Of these, the quinalizarin procedure ( 1 ) appeared to be reported most frequently. Although this method is capable of giving good results by visual comparison of the colors under certain conditions, i t is of limited value when the color intensities are measured in a spectrophotometer. Weinberg ( 1 2 ) and Ellis ( 4 ) account for this situation by noting t h a t the differences between the absorption spectra of quinalizarin and the boronquinalizarin complex are very small at nearly all wave lengths. In other words, because of the overlapping spectra of the reagent and the complex, the sensitivity a t a given wave length is poor.
The eye, on the other hand, is a+le to integrate all the small differences over the entire visible spectrum and can judge small color and, therefore, small concentration differences. The method reported by Hatcher and Wdcox ( 6 ) )using carmine S o . 40 N.F. as the colorimetric reagent for boric acid, seemed to satisfy best the requirements for a method which is rapid, accurate, relatively unaffected by foreign ions, and reproducible over the desired range of boron concentrations. The original method of Hatcher and Wilcox was proposed for use on plant materials and natural n-ater, but in adapting i t for use in this study the recommended procedure for destroying organic matter-viz., dry ashing with calcium oxide-was too timeconsuming when used for biological fluids and tissue. Wet ashing with concentrated sulfuric acid and hydrogen peroxide has been reported by Hoffman and Lundell ( 7 ) to give low resiilts owing to volatilization of boron. This was confirmed experimentally by the writers. Two other methods for destroj-ing organic matter were investigated. These were: dry ashing with a mixture of sodium and potassium carbonates, and dry ashing ivith lithium carbonate a t 650' C. The latter was the most convenient of the methods studied, and was used in the procedure described below.. After the fusion of the biological material with lithium carbonate, the melt was dissolved in hydrochloric acid. A faint yellon-ish turbidity which developed interfered with the measurement of color intensity. T o eliminate the difficulty, the eolution obtained by dissolving the melt was centrifuged, and a standard volume of the clear supernatant liquor was used for analysis. S o boron could be detected in the precipitate Separated by this treatment. Another critical point in the procedure was found to be the acid concentration required for color development. Satisfactory results were obtained, provided the concentration of sulfuric acid exceeded 90%. Even after the development of the color, precautions were necessary to prevent dilution of the sample by atmoqpheric moisture. As reported by Hatcher and Kilcox ( 6 ) , both carmine S o . 40 N.F. and carminic acid ivere satisfactory over most of the range of concentrations investigated. I n the present study, the color developed much more rapidly when carminic acid was used. The maximum intensity was reached in tubes stored a t room temperature (24' to 2 i " C.) Bithin 5 minutes after the addition of the reagent, and the eo101 \vas stable for a t least TO minutes. On the other hand, when carminc S o . 40 S.F. \vas used, the time required to reach the optimum color n a s 35 minutes (see curve', Figure 1). METHOD
Apparatus and Reagents. The Beckman DU Spectrophotonieter was used Kith Cores cells of a I-em. light path for the absorption measurements. Corning alkali-resistant (low-boron) glass bottles and apparatus, platinum crucibles, and polyethylene sample containers were used viherever possible to minimize chances of boron contamination. Sulfuric Acid, concentrated, analytical reagent grade. J. T. Baker's reagent grade was found to be substantially boron-frre and is recommended. Carminic .icid Reaqent w i s prepared by dissolving 250 mg. of carminic acid C . P . (Fisher Scientific Co. S o . A-93) in concentrated sulfuric acid and diluting to 1 liter Kith concentrated sulfuric acid. Aqueous 6 S Hydrochloric Acid. Lithium Carbonate, anhydrous, analytical reagent grade. J. T. Baker's reagent grade was found to be subst,antially boronfree and is recommended. Preparation of Standard Solutions. Boron stock solution was prepared by dissolving 0.2203 gram of Sational Bureau of Standard sodium tetraborate decahydrate in distilled water and diluting to 250 ml. The boron content of the concentrated solution was checked using the A.O.A.C. identical p H titration procedure (1 ml. is equivalent to 100 y of boron). Suitable diluted solutions may be made from this solution by diluting measured portions with distilled I n t e r .
V O L U M E 2 7 , NO. 2, F E B R U A R Y 1 9 5 5 Table I.
2S7
Calibration Summary
h-o. of Accuracy Material Determinations Calibration Curve Limits" H u m a n blood 19 H u m a n urine 21 B = 56.04 11.0 R a b b i t blood 19 Animal tissue 21 B = 55,Od - 0 . 5 r0.8 B . Alicrograms of boric acid fae boroui. A . Absorbance reading a t 575 m k . Accuracy limits ( 2 X standard error of estimate) will include about 95% of the results.
PROCEDURE
-40.10-gram sample of lithium carbonate is weighed into a clean platinum crucible. Two milliliters of the fluid (or an appropriate weight of tissue sample) containing not more than 30 y of boron are transferred into the crucible and, if necessary, excess water is removed by evaporation on the steam bath. The crucible is placed in a muffle furnace and heated for 15 minutes a t 100" C., after which the temperature is raised gradually to 650" C., and the heating is continued for 1.5 hours or until the sample is free from carbon. The sample is removed from t h e muffle furnace and allowed t o cool to room temperature, and 2.00 ml. of 6,V hydrochloric acid are pipetted into the crucible. The liquid is mixed well by swirling and poured into a 15-ml. centrifuge tube, and the contents are centrifuged a t 3000 r.p.m. for 2 minutes or until the supernatant liquor is clear. 040
0 35
I
I
'
I
I
CALIBRATION
! I
I
I
through the procedure. The writers had no difficulty in finding human blood which was boron-free. The blood was checked bv ashing a suitable portion in the presence of lithium carbonate and e x w i n i n g the ash with a large quartz spectrograph. KO boron lines could be detected on the plates under these conditions, b u t when 1 p.p.m. of boron was added to the same blood, and the resulting mixture checked in the same manner, the boron lines were easily detected. Urine, on the other hand, frequently contained trace amounts of boron, which were removed as the volatile methyl borate according to the procedure described by Chapin (f3). The residual material, when made u p to its original volume, \vas then used for the preparation of standards. Animal tissue samples used for calibration studies were also treated by the Chapin technique, when necessary, to remove boric acid. The absorbance readings a t 575 mM nere recorded for the corresponding amounts of boron used and, using prescribed statistical techniques, a calibration curve of boron content on absorbance \$-ascomputed. There was no significant difference between the calibration curves obtained for various samples of human and rabbit hlood and urine. The calibration curve for a varietv of animal tissues, however, differed slightly but significantly from the blood and urine curve (Figure 2). These calibration curves are given in Table I along with the accuracy limits [twice the standard error of estimate ( I S ) ] and the number of determinations performed for obtaining the data.
I
CURVES I
I
DISCUSSION
Previous workers (6)have reported that nitrates and nitrites were the ions which caused the most serious interferences, but the effects of these are minimized in the procedure by the addition of hydrochloric acid. No other inorganic substances commonly found in biological materials seemed to interfere significantly with the method. If the boron content of the sample exceeds 30 y, the proper aliquot should be taken to keep the boron content \Tithin the range of the method. I t was found that the following materials could be analyzed satisfactorily by this procedure: blood, urine, lung, heart, thymus, liver, spleen, stomach, duodenum, kidney, prostate or uterus, brain, voluntary muscle, skin, urinary bladder, jejunum, cecum, and terminal colon, as well as fecal matter and gastiic contents. LITERATURE CITED
Figure 2
One milliliter of the liquid is transferred into a 100 X 20 nim. a1l;ali-resistant glass test tube (boron-free), 5.00 nil. of con( trated sulfuric arid are added, followed by 5.00 nil. of the minir acid reagent, and the tube is covered to exclude atmospheric moisture. (Type 11.Parafilm was found to be excellent for this purpose. It may be purchased from Para Laboratory Supply Co.. Trenton, S . ,J.) The tube is shaken well, and allowed to stand for a t least 5 minutes at' room temperature to develop the color, and the absorbance of the solution is measured with a Beckman DU spectrophotometer in a 1-cm. covered cell a t 5 i 5 mM. .i blank sample of similar material from which the boron has been removed, if necessary, by the Chapin ( I S ) distillation, should be carried through the procedure with each set of samples. The boron content of the solution is determined by reference t o a standard curve made by carrying the biological material containing knonm quantities of boron through the above procedure.
STANDARDIZATIONOF
METHOD
The method was standardized by adding known amounts of standard boron solution to 2.0-ml. (or 2.0-gram) boron-free samples of blood, urine, and tissue, which in turn were carried
Assoc. Offic. Agr. Chemists, "Offi&l llethods of .lnalyais." i t h e d . , p. 117, 1950. Berger, K . C..and Truog, E.. S o i l . Sei., 57, 25 (1944). Dermott, W., and Trinder, S . . J . 9 g r . Sci.,37, 15%5 (1947). Ellis. G . H., Zook, E. G., and Baudisrh. O., A x . ~ L . CHEXI.. 21.1345 il949). E'izzotti, C., and Seluii. I,., CiiimI'ca e indicslria (JJilmi), 3 4 , 265-6 (1952). Hatcher, J. T.. and \Tilcox. L. I-.,.4su.. CHEW.,22, 507-~69 ( 1950) . Hoffman. ,J. I.. and Lundell, G . E . F., J . Rcsrarcii .\-at/. R c r v . S/enrlarrls, 22,465-70 (1939). IIelvin, E. H.. and O'Connor, R. T., h s . \ r . . CHEM.,13, 520-4 (1941). Smith. F. AI., Svhrenk, IV. G . . and King, H. H.. I h i d . , 2 0 , 941-3 (1948).
Steadman, L. T., private communication. Trinder, S . , Analyst, 73, 494-7 (1948). Weinberg, S., Proctor, K . L., and lIi1ner. .is
