V O L U M E 20, NO. 1, J A N U A R Y 1 9 4 8
73 ~~
percentages of lime and silica remain unknown, and only the ratio of these percentages is used. 4CCURSCY 4ND SPEED
The general reproducibility of results is approximately 10% of the lime-silica ratio value. Table I shows a comparison of results obtained on eleven slags run spectrographically and chemically. The samples were run in quadruplicate ipectrographically and all results tabulated. Ordinarily, sample? are run in duplicate. Occasionally, slags are found where repeated check-, give variatjons of 1.0 or even 1.5 in the ratio. These same slags, after once having been powdered, have a tendency to cake upon long standing. Their true lime-silica ratio is generally over 3.0. Both the caking and erratic results, it is believed, can be accounted for by the presence of relatively large amounts of freeunreacted lime. Free lime causes the spectrographic result to run high. To date, attempts to overcome this difficulty have been unsuccessful.
Table I.
Comparison of Linie-Silica Ratios
Slag
Chemical
A B
0.67 0.76 1.85
C
D
E F G H I J
~
Spectrographic Individual
Average 0.67 0.68 1.9
1.9
2.04 2.27
2.3
2.91
3.0
3.70
3.6 4.8
8.43
8.5
4.70 6.00
6.1
If there are no interruptions, a lime-silica ratio can be detcrmined within 15 to 20 minutes of the time of delivery of the powdered sample to the spectrographic laboratory. RECEIVED June 4,1947.
Spectrographic Determination of Beryllium in Biological Material and in Air JACOB CHOLAK AND DONALD M . HUBBARD Kettering Laboratory of Applied Physiology, University of Cincinnati, Cincinnati, Ohio
A spectrographic method for the determination of beryllium in biological material and in the air has been developed, which is capable of detecting as little as 0.25 microgram of beryllium. Details are given for the analysis of samples of freshly voided urine without ashing and for a wet-ashing procedure w-hich is generally applicable. Rlethods for collecting and analyzing air samples are also given. The most satisfactory procedure consists in asbing the samples with sulfuric, nitric, and perchloric acids, isolating the beryllium as the phosphate, and dissolving the phosphate precipitate in an acid spectroscopic buf-
B
ERYLLIUII has been determined in alloys, ores, and rocks colorimetrically ( 5 , 9, 11, 16), fluorometrically (6, 15, 17, 18), and spectrographically (4, 8, IO, 12, I S ) . Hyslop and coworkers (7’) found that most of these methods lack sensitivity when applied to biological material, and that it was necessary to evolve special methods for the determination of beryllium in animal tissues. As a result, they investigated three possible tests (71, but found that none of these was uniformly applicable. The principal difficulty in the use of chemical methods is their lack of specificity and the consequent need for removing interfering ions (6, 14). Since many interfering ions (iron, calcium, magnesium, and phosphate) occur regularly in biological material, the difficulty of isolating minute amounts of beryllium thercfrom without appreciable loss is very serious. Accordingly, it has seemed desirable to investigate the applicability of spectrographic procedures as a means of studying the behavior of beryllium in the animal organism. The results, of preliminary spectrographic examinations of solutions of pure beryllium sulfate were so promising from the aspects of specificity and sensitivity as t o justify further investigation of the entire anaIytica1 procedure. I t was found earIy that the spectrographic techniques themselves offered no difficulties and that the most important factor in the reliable esti-
fer solution. Aliquot portions (0.2 nil.) of the latter solutions are used to impregnate graphite rods which are used as the lower positive electrodes of direct current arcs. Densitometry involves the plotting of H and D curves obtained for the stepped line of beryllium at 2348.6 hi. and that of the internal standard, thallium at 2379.7 A,, and obtaining the separation of the two curves at a constant blackening ( T = 0.50). This separation is then evaluated from a working chart prepared beforehand from known amounts of beryllium which are added to the spectroscopic buffer solution.
mation of minute quantities of beryllium related to the preparation of samples. The latter involved the destruction of organic matter and the isolation and concentration of beryllium without loss, points which have not been studied by others. Procedures for both preparation and analysis of samples have been dcveloped which are applicable t o a wide variety of materials, including those which are usually encountered in physiological and environmental investigations. SPECIAL RE:%GENTS
Calcium Phosphate Solution. Two and one-half grams of calcium carbonate (calcite) are dissolved in just sufficient ooncentrated hydrochloric acid (specific gravity 1.19) to effect sol? tion (about 7 ml.). Diammonium phosphate (2.5 grams) IS added and the solution is made up to 100 ml. with distilled water. If a precipitate forms, it is resolved by adding hydrochloric acid drop by drop. Spectroscopic Buffer Solution. Any ionizable salt or salt mixture may be used. The following solution was used because of its availability in connection x i t h other spectrographic work: 125 ml. of a stock solution ( l ) ,designed to simulate urine in Its salt content (sodium chloride, 170.5 grams; potassium chloride, 63.5 grams; calcium chloride hexahydrate 31.5 grams; magnesium chloride hexahydrate, 20 grams; and sodium dihydrogen phosphate monohydrate, 37.5 grams, dissolved in and diluted to 1000 ml. with 10% by volume hydrochloric acid) are mixed with
74
ANALYTICAL CHEMISTRY
50 ml. of hydrochloric acid, specific gravity 1.19, and 5 ml. of thallic nitrate solution (1 ml. = 10 mg. of thallium), and the whole is diluted to 500 ml.
determined by experience and by anticipation of the quantities of beryllium available. Samples Obtained by Impinger. The liquid (water or alcohol) is transferred from the impinger to a 250-ml. Pyrex beaker, and the procedure prescribed for samples obtained by electrostatic precipitation is followed.
PREPARATION OF SAMPLES
Urine (Freshly Voided). Forty milliliters of urine arc placed in a 50-ml. centrifuge tube having a round bottom, 0.5 ml. of the calcium phosphate solution is added, and then small drops of concentrated ammonia (specific gravity 0.900) are added until the solution is just alkaline to phenol red. The solution is ccntrifuged for 3 minutes a t 2500 to 3000 r.p,m. and the supernatant liquid is decanted and discarded. The precipitate is dissolved in 0.5 ml. of hydrochloric acid and a little distilled water and the solution is transferred quantitatively to a graduated 15-ml. conical centrifuge tube. One milliliter of the spectroscopic buffer solution is added and the volume is adjusted to 1 ml. by evaporating the solution in an oil or glycerol bath a t about 120" to 140" C. Animal Tissues. Ten grams or more of tissue or blood (or 50 ml. of urine) are placed in a 250-ml. Pyrex beaker, 5 ml. of concentrated sulfuric acid and 20 ml. of concentrated nitric acid are added, and the mixture is heated until charring occurs. The char is destroyed by the addition of a small amount of nitric acid and heat, and the process is repeated until no charring occurs when the mixture is heated until fumes of sulfur trioxide arc evolved. The digestion and oxidation may be hastened if, toward the end, 5 ml. of perchloric acid (70 to 727,) are added. Aftei oxidation is complete, all but a trace of the sulfuric acid is evaporated off. On cooling, the solution is transferred to a 50-ml. centrifuge tube and dealt with exactly as was the sample of freshly voided urine. I n digesting samples of bone or other samples where large amounts of calcium are thrown down as the sulfate, the calcium sulfate is removed by filtration or centrifuging before theberyllium is isolated as the phosphate. Because of the large amount of ammonium sulfate which is formed, "salting out" may occur before a final volume of 1 ml. can be reached. When this occurs, 2 ml. of spectroscopic buffer are used, and the final volume is adjusted to 2 ml.
SPECTROGRAPHIC TECHNIQUE
Graphite electrodes (0.78 em. in diameter and 3.75 em. in length, prepared to have a crater 3 mm. in diameter by 10 mm. in depth) are impregnated with 0.2-ml. portions of the solutions which have resulted from the preparatory procedures. Duplicate rods are used for each solution. The impregnated rods are dried for 30 minutes a t 100" t o 110" C. and then are used as (lowe1j positive electrodes of the arc. The (upper) negative electrodes are made up of fresh 5.00- to 7.5-cm. lengths of rod, oneknd of which has been turned to a fine point in a pencil sharpener. EYcitation is accomplished by direct current from a 110-volt direct current power line, the arc being operated at 10 amperes. Earh exposure continues for 2 minutes, and the spectra are photographed on Eastman KO.33 plates. Each spectrum is "stepped" by means of a stepped sector (factor 2) which is rotated before the slit (0.03 mm. wide and 15 mni. long) of a Bausch & Lomb large Littrow quartz spectrograph. After the plates have been developed in D-19 developer, fixed, washed, and dried, th,e H and L) curves are obtained forothe beryllium line a t 2348.6 A. and the thallium line at 2379.7 A , , by measurement with a nonrecordirig Table I.
Beryllium Recovered from Urine by Direct Method of -4nalysis (No Ashing) Be Added. 7 0.0
(40-ml. samples)
Be Found,
y
0,o
O.J.O.5
0.65, 1.10, 0.60 1.05
8.0,8.0
2.0,z.o 3 . 5 , 3 ,h 8.8,g.O
1.0,l.O 2.0,z.o 4.0,4.0
ALTERNATE METHOD FOR BLOOD WITH IRON REMOVAL
Table 11. Beryllium Recovered Following Addition to Biological Material
After 10 grams or more of blood have been digested according to the foregoing procedure, the beryllium is isolated as the phosphate by the method given for dealing with freshly voided urine. The phosphate precipitate is then dissolved in hydrochloric acid and the solution is transferred to a separatory funnel. The volume of the solution is adjusted to 20 ml. with distilled water and iron is removed by the following procedure.
(Materials ashed by n e t method) Be Found, y Be Added, y 0,o 080 0.5,0.5 0.4,0.4 1.0,l.O 1.0,o.g 1.9,z.o 2.0,z.o 4.0,4.0,4.0 3.7,3.7,4.4 0, o a 10 grams of liver 0,o 0.5,0.5 0.8,O. 60 10.0,10.0 11.5,0.00 0,o 0,o 10 grants of blood O..i, 0.5, 0.5, 0.a 0,65,0.60,0.60,0 5 1.5.1.6 1.6.1.6 0 Iron not removed.
Four milliliters of a 6% by weight cupferron solution are added and the mixture is shaken with successive 10-ml. portions of chloroform until the last portion of chloroform added is found to be colorless. The chloroform lavers are dis-
6-
-
tion which had been added. DEPOSITED DUST OR SAMPLES OF DUST OBTAINED FROM AIR
solution is next added and the mixture is evaporated in the oil bath to the volume of buffer solution which !vasa dded. The amount of buffer solution to be added is determined by experience
Material 50 mi. of urine
P3
't
02
-
I
V O L U M E 20, NO. 1, J A N U A R Y 1 9 4 8 densitometer ( 2 ) . The average separation between the two H and D curves a t a constant blackening value (at T = 0.50) is then read from a calibration curve (Figure 1) prepared by adding known amounts of beryllium to 40 ml. of distilled water and proceeding according to the method given for t'he preparation of freshlyvoided samples of urine ( 2 ) . RESULTS
The extent of the recovery of known amounts of beryllium added to freshly voided urine and handled without ashing is shown in Table I. The recovery achieved following the use of wet-ashing techniques is given in Tables I1 and IV. DISCUSSION
The most accurate analytical range of the worlting curve is that which lies between 0.25 and 2 micrograms per ml. of solution. Larger quantities of beryllium may be determined by diluting the prepared sample with suitable quantities of the spectroscopic buffer. Thus if the quantity to be read from the curve exceeds 2 micrograms, it is advisable to repeat the analysis following dilution with spectroscopic buffer solut,ion. The inaccuracy above 2 micrograms per ml. is due to the varying degrees of self-reversal shown by the beryllium line a t 2348.6 A, under the conditions of excitation. This effect is illustrated in Figure 1, in which each cross on the figure represents the average separat,ion value obtained for a set of six spect,ra on a single plate. The straight line passing t,hrough the circles is the working curve, and a t each concentration the circle gives the mean separation value obtained from the total number of spectra taken. Although data for 8 micrograms per ml. are not shown, the few plat,es taken a t this concentration show even wider variations in blackening curve separation values than do those represent,ing 4 micrograms per ml. The amount of self-reversal obtained depends upon the concentration of the beryllium vapor in t,he arc. This concentration of vapor varies within limits set by the absolute amount of beryllium present and t'he width of the gap betvieen the electrodes each time the arc is st,ruck. Since for the authors' purposes the Ividth of this gap, approximately 5 mm., \Tas judgpd visually, variat'ions up to 50% may well have occurred. I t is very likely that more consistent blackening curve separation values for concentrations exceeding 2 micrograms per nil. can be obtained by maintenance of a constant, gap between the electrodes. This can be done by projecting an image of the arc upon a screen, and by constant'ly adjust,ing the e l ~ c trodes so as to maintain their images a t a fixed distanrc. apart, as indicated by marks upon the screen. Experience with spect'rographic methods of analysis has showi~ that the method of densitometry based on the data of blackening curve separations ( 2 ) is the most practical when biological material is handled. Except for extremely l o concentrations ~ of sonie mctal.;, there is no need for calibrat,ion of plates or corrections of the background ( 3 ) , while there is added advantage in the fact that the procedure frequently permit,s the derivation of a single calibrat,ion curve for the entire workable range. This is not possible when the intensity ratio met,iiod is employed; it is necessary, usually, to derive several working charts to rover the analytical range even if stepped spectra are used. The accuracy of the intensity ratio method is also dependent, upon the need for continual calibration of plates and to some degree upon corrcctions of background. Direct ashing of biological material in either silica or platinum dishes, at temperatures as low as 500" C., results in significant losses of beryllium. Such losses are illustrated in Table 111. The method for the determination of beryllium in urine has been designed to handle samples of freshly voided urine without ashing. Aged urine samples, in which deposits of phosphate have formed, may also be handled by this technique, but only after the deposit has been dissolved by t,he addition of acid. The act'ual volume of the urine which is subjected to analysis in these instances will be less than 40 nil., and the dilution fartor may
7s affect the sensitivity of detection. Since wet ashing permits the use of larger aliquots, it is preferred for handling aged samples of urine. Certain of the beryllium compounds which may be encountered in the air are very inert and can be dissolved only after the employment of a fusion technique. Such a procedure is time-consuming and tedious, and, in view of the data of Table 111, probably unsatisfactory However, it was found possible to deal with such materials satisfactorily by dispersing sediment or particulate matter in the final solution just before impregnating the rod. This can be done by blowing into the solution through the pipet which is used to impregnate the electrodes. This procedure was tested with a suspension of beryllium oxide, 3 ml. of which, corresponding to 15 micrograms of beryllium as the oxide, were pipetted upon 12.5-cm. Whatman No. 40 filter papers in triplicate. The papers were digested and the beryllium was isolated in 15 ml. of spectroscopic buffer solution, 15, 15, and 16 micrograms of beryllium, respectively, being recovered. The beryllium and thallium lines used in this work may be masked by iron lines if the instrument employed does not have adequate dispersion or resolution. I n spectra taken with the Bausch & Lomb quartz large Littrow spectrograph, no difficulty was encountered in identifying the beryllium line in the presence of large amounts of iron. The densitometry of weak beryllium lines, however, was affected, in that the presence of large amounts of iron apparently increased the intensity of the beryllium line to a greater extent than that of the internal standard. The effect of iron is illustrated in Table IV, in which it may be Seen that the recoveries of small quantities of beryllium are in closer agreement with the added quantities when iron has been removed according to the procedure of the alternate method. The error of analysis in this range in the presence of iron, while large on a percentage basis, is not, however, too significant when it is examined on the basis of the absolute amounts of beryllium involved. When the quantities of beryllium are above 2 micrograms per nil. of solution, iron offeIs no difficulties, since dilution with the spectroscopic buffer solution reduces the iron concentration to a level a t xyhich it has very little effect upon the intcn5ities of the beryllium and thallium lines. No effect has been noted when the iron concentration in the final solution does not excred 1 mg. per ml. I n the authors' hands, iron x a s removed only from samples of blood and large samples of other tissue when it was expected that the beryllium would have to be concentrated in a volume of 1 ml. This procedure \\-as followed rather than that of correcting for the iron by the derivation of a separate calibration curve from a standard solution containing a fixed amount of iron. The use of such a correction procedure wa, discarded \Then it ITas found that the iron had to be removed i n order to maintain adequatr control of its effect3 within the
Table 111.
Type of Dish Glazed silica Glazed silica Glazed silica Glazed silica Platinum Platinum
Table IV.
Loss of Beryllium issociated w i t h Dry ishing at 500"
c.
JIate'rial Urine (100 ml.) Urine (100 ml.) Urine (100 ml.) Urine (100 m1.j Urine (60 ml.) Urine (60 ml.)
Be Added,
Be Recovered,
Y
Y
1, 1 2, 2 , 2
0.9, 0 . 4 0 . 4 , 1 . 2 , 1.4
4,4 16, 16 4
9.6,2.4 0.5
8
3.2
3.0,2.2
.Iverage Recovery,
% 65 50 65 38 12.5 40
Recovery of Beryllium in Presence or Absence of Iron
(Material = 10 grains of blood except a s indicated) Micrograms of Be Recovered Fe present Fe removed
Be Added, y
0, 0 0.5 0 . 5 (10 grama liver) 1.5
0,o
1 . 4 , 0.90, 0 . 8 , 0 . 7 0 0.8,0.6
1.7,l.g
0, 0 0.6~,0.60,0.60,0.50 0.5 1.60, 1.60
ANALYTICAL CHEMISTRY
76 arc. Moreover, contrary to expectations, the presence of iron did not increase the sensitivity of detection of beryllium, since the lower limit of detectability (0.04 microgram on the arc) remained unchanged in its absence. LITERATURE CITED
(1) Cholak, J., IND.ENG.CHEY.,ANIL. ED.,7,287 (1935). (2) Cholak, J., and Story, R. V., J . Optical Soc. Am., 31,730 (1941). (3) Ibid., 32,502(1942). (4) Fesefeldt, H., 2.physik. Chem., Abt. A,140,254(1929). (5) Fischer, Hellmut, Wiss. VerOfentl. Siemens-Konzern, 5, 99 (1926). ( 6 ) Fletcher, M . H., White, C. E., and Sheftel, M. S., IND.ENG. CHEM., ANAL. ED.,18,179(1946). (7) Hyslop, F.,Palmes, E. D., Alford. IT. C., Monaco, A. R., and Fairhall, L. T., Div. Ind. Hyg., Natl. Inst. Health, Bull. 181, (1943).
(8) Jaycox, E.K., J . Optical Soc. A m . , 35, 175 (1945). (9) Kolthoff, I. M., J . Am. Chem. Soc., 50,393(1928). (10) Masi, O.,Spectrochim. Acta, 1, 501 (1941). (11) Osborn, G. H., and Stross, W., Metallurgia, 30, No. 175, 3-6 (1944). (12) Peer, K.C., Light Metal Age, 1, No.4,14,22(1943). (13) Pieruccini, R.,Spectrochim. Acta, 2,269 (1943). (14) Sandell, E. B.,"Colorimetric Methods for the Determination of Traces of Metals," New York, Interscience Publishers, 1944. (15) Sandell, E.B.,IND. ENG.CHEM.,A N A L .ED., 12,762 (1940). (16) Stross, W., and Osborn, G. H., J . SOC. Chem. I n d . , 63, 249 (1944). Ricerca sci., 13,726 (1942). (17) Venturello, G., (18) White, C. E., and Lowe, C. S., IND. ENG.CHEM., A N A L .ED., 13, 809 (1941). RECEIVED July 7 , 1 9 4 7
Determining Small Amounts of Calcium in Plant Materials A Colorimetric Method EDWARD H. TYSER, West Virginia Agricultural Ezperirnent Station, Morgantown, W. Vu. A rapid photocolorimetric method for the determination of small amounts (0.4 to 1.1 mg.) of calcium in plant materials is based on the measurement of color diminution that accompanies precipitation of calcium chloranilate from intensely colored chloranilic acid solutions. Factors affecting transmittance of chloranilic acid solutions and tolerable interfering cation concentrations are re-
C
ALCIUM is important in the nutrition of practically all green plants. At present, it is determined almost exclusively by gravimetric and volumetric procedures after precipitation as calcium oxalate. Toe (8) describes two methods for the colorimetric determination of calcium-the alizarin and the oleate procedures-neither of which, however, is \vel1 known or suited for rapid routine determinations. Barreto (d) has recently noted the insolubility of the calcium salt of chloranilic acid and has developed gravimetric and colorimetric procedures for the determination of calcium in soil extracts based on this insolubility. The gravimetric procedure would appear to have no practical advantage over present oxalate methods. The colorimetric procedure, however, might have considerable value. Iinfortunately, Barreto gives no clue as t o the precision or accuracy of either procedure. The colorimetric procedure is dependent upon the color diminution that accompanies the precipitation of calcium chloranilate from intensely colored solutions of chlomnilic acid. The procedure given is essentially as follows:
ported. Iron, aluminum, barium, strontium, sodium, potassium, and manganese do not interfere in amounts normally encountered in plants. The most serious interference arises from occlusion of magnesium. The minimization of this error is discussed. The colorimetric method compares favorably in precision and accuracy as a routine procedure with an official volumetric macroprocedure.
developing a photocolorimetric method for the rapid determination of calcium in plant materials. PROPERTIES AND REACTIONS OF CHLORANILIC ACID
Chloranilic acid (2,5-dichloro-3,6-dihydroxyquinone)has a molecular weight of 208.99 and is a strong dibasic organic acid. I t dissolves sparingly in water to give intensely colored solutions very similar in color to that of potassium permanganate. The calcium salt is reddish-purple, crystalline, and easily filterable.
An acid extract of soil is evaporated to dryness, redissolved in distilled water, alkalinized with ammonia, made up to 200-ml. volume, and filtered. One hundred milliliters of the filtrate are concentrated to 30 ml. To the concentrate, 100 ml. of 0.15% chloranilic acid are added. The solut,ion is then slightly acidulated with hydrochloric acid and brought up to 200-ml. volume by the addition of distilled water. After 2 hours of standing it is filtered. The filtrate is compared with equally acidified chloranilic acid standard solutions. The present study was made t o determine the effect of hydrogen-ion concent,ration and temperature on color intensity, time required for maximum color diminution, and sensitivity to small amounts of calcium and interfering cations with the object of
Figure 1. Spectral Transmittance Characteristics of Chloranilic Acid
