I n addition to the interference caused by the metallic ions, water causes about 0.1% decrease in the niobiuni-hydroquinone color for every 1 mg. present in 10 ml. of solution; phosphate causes about 0.07% decrease per mg.; but fluoride, added as potassium fluoride, has no observable effect. It is obvious that reagents used in this procedure should be free of phosphate and protected from atmospheric moisture.
Table IV. Results for Niobium in Uranium-Titanium-Niobium Alloys
I
0.487 0.470 0.490 0 .46ga 0 . 466n 0 . 500a 0.480 f 0.014 1.02 1.00
0.469 0.465 0.469O 0.4744 0,477a
RELIABIL\TY
KO standard samples of plutonium or uranium alloys containing niobium were available. Therefore, the reliability of the method is based upon the analytical results for solutions containing known amounts of niobium and upon the reproducibility of results for uraniunititanium-niobium alloys. The solutions were fumed with sulfuric acid and treated as solutions of samples; the results should indicate the reliability for actual samples. Any precipitate that formed during fuming was transferred quantitatively to the test tube and the entire mixture was extracted. Data for 38 representative determinations, as giren in Table 111, show an
0.5
I1
Av. 0.471 & 0.005b 1.0 1.01 1.02
1.02 1.00
1.01
1.01
Av. 1.01 f 0.01
I11
0.5
0.258 0.2.58 .~
.
0.250 0.2E16~
0.243~ Av. 0.253 5
0.006
_
1.01 =I= 0.01 0.240 0.2615 0 .256a 0.253 0.252 0.252 =I= 0.008
Double extraction performed. Standard deviation is precision index used for all results. a b
average of 99.9% for the niobium found, with a standard deviation of 2.0y0. The niobium in the alloy samples, which contained 0.25 to 1.00% of niobium and titanium, was determined by two analysts. Although the titanium concentrations were low, double extractions were performed with some of the samples to test the method. As shown in Table IV, the results for the three representative samples obtained by the two analysts agree within 2% and have a precision of 1 to 3%. The time required for analysis depends upon the ease of solution of the sample. After the samples are dissolved, 15 to 20 determinations may be made in 1 day. LITERATURE CITED
(1) Ikenberry, L., Martin, J. L., Boyer, W.J., ANAL.CHEM.2 5 , 1340 (1953). ( 2 ) Stevenson, P. C., Hicks, H. G., Ibid., 25,1517 (1953). (3) Waterbury, G. R., Bricker, C. E., Ibid., 29, 129 (1957). RECEIVED for review September 16, 1957. Accepted January 8, 1958. Work done under the auspices of the U.S. Atomic Energy Commission.
Determination of Microgram Quantities of Fluoride H. M. NIELSEN Utah Stafe University, logan, Utah The determination of trace amounts of fluoride in the quantities found in some animal tissues is difficult, especially when the sample size must b e restricted. By employing an ion exchange technique for concentrating the fluoride and freeing the solution from interfering ions, 1 - to 10-7 quantities can b e estimated with a precision within 5% of the fluoride present.
S
papers have reported methods for determining fluoride in small amounts. Usually the sample is isolated from interfering substances by the Willard-Winter (IO) distillation or by some modification of this procedure. The isolated fluoride has been estimated, apparently with satisfactory results, by titration (8-4, 8), fluorimetry (6, 9). and spectrophotometry (2, 3, 7 ) , if the fluoride concentration is not too low. In the isolation of fluoride by the distillation procedure, rather large amounts of distillate must be collected to ensure quantitative recovery; the concentration may thus be too low for satisfactory estimation when certain substances are analyzed. Concentration of the distillate by EVERAL
evaporation usually is unsatisfactory because interfering substances present in small amounts may be concentrated to a point where they exert a significant effect. Making the solution basic to prevent loss of fluoride and subsequently adjusting to the proper p H for measurement add to the total salt concentration of the sample, a factor which must be controlled when very small amounts of fluoride are being det ermined. The method reported here employs an ion exchange resin for concentrating the fluoride. Estimation is by a modification of the spectrophotometric method of Megregian ( 3 ) . Attebury and Boyd ( 1 ) first showed that the halides could be absorbed on an anionic exchange resin, from which they could then be separately eluted. They used the strongly basic resin, Dowex 2, in the nitrate form. Nielsen and Dangerfield (6) applied this principle to the determination of atmospheric fluoride, using Duolite A41 in the hydroxyl form. The hydroxyl forms of both Duolite and Dowex 1 and 2 failed to give results of sufficient precision for the author’s purposes, so a more suitable
form was sought. Dowex 1 in the acetate form has been used in the laboratory for the past year and has given satisfactory results in determining fluorides in 0- to 10--y amounts in volumes of as much as 250 ml. APPARATUS A N D REAGENTS
Beckman Model DU or B spectrophotometer or equivalent instrument. 10-mm. matched cuvettes. Anion exchange resin, Dowex 1-X8, 200 to 400 mesh. Sodium acetate solution (1M) in distilled water. Sodium acetate solutions ( O . l M , 0.2 144, and 0.3M) made by diluting the 1 M acetate solution. Standard fluoride solution, 10 y per ml. Reagent A, 1.000 gram of Eriochrome Cyanine R (Geigy) dissolved in distilled water and diluted to 1 liter. Reagent B, 0.175 gram of zirconyl nitrate dihydrate, dissolved in 500 ml. of distilled water, t o which are added 500 ml. of concentrated hydrochloric acid (reagent grade, specific gravity 1.19). Reference solution (for setting the zero point on the spectrophotometer). To 105 ml. of distilled water are added 10 ml. of Reagent A and 5 ml. of concentrated hydrochloric acid. VOL. 30. NO. 5, M A Y 1958
1009
Table 1.
Precision before and after Concentration on Column
Coeff. of Variation,
Aliquot,
s o . of
Ail. 5
50
Detns. 6 9
Table II.
Recovery of Fluoride from Distillate and Undistilled Solution
Fluoride, y Added Found 0.2 0.17 2.0 2.02
s o . of
Std. Dev. 0.123 0.083
Fluoride, y Added Found 1.0 0.99
%
72.4 4.6
Std. Dev. 0.117
Detns. Original 12 solution Concd. from 6 1.0 0.95 0.176 distillate Distillate 6 0.1 0 . 1 (one sarnp1e)a direct a Absorbance of other five samples indicated no fluoride present. These data show the unreliability of a direct determination on distillates a t this concentration. Table 111. s o . of
Recovery of Constant Amount of Fluoride Added to Variable Amounts o f Kidney Tissue Wt. of
Detns. 5 3
5
Tissue, Grams 2.5
Fluoride,
Added
5.0
10.0
Ion exchange columns, straight borosilicate glass tubes, 9-mni. inside diameter and approximately 12 inches long. A 00, one-hole rubber stopper containing a piece of 5- or 6-mm. glass tubing 4 or 5 em. long is covered with a circular piece of thin nylon cloth, slightly larger than the smaller diameter of the stopper. This is inserted into the tube to contain the resin. KO portion of the cloth should extend beyond the end of the tube. A portion of resin in the chloride form is suspended in distilled water and allowed t o settle, and the fines are decanted. The resin slurry is added to the tube to make a column when settled 2 em. in height. After the water has drained off and the surface is firm and smooth, a small plug of glass n.001 is inserted to prevent distortion of the surface when liquid is added t o the tube. The resin is converted to the acetate form by dripping into the column (4 to 12 drops per minute) 25 ml. of 1iM sodium acetate. A small amount of chloride remaining in the resin does no harm. The resin sn-ells to a height of about 2.5 cm. when converted to the acetate form. The acetate solutions and samples are conveniently added t o the columns by means of pipets. The suction end of each pipet is equipped with a short length of gum rubber tubing and a screw clamp. The pipet is supported above the column and the solution is allowed to drip onto the resin. A small wire (26-gage Nichrome) inserted in the tubing aids greatly in controlling the rate of flow. Excess acetate is removed from the resin by filling the tube with distilled water and allowing it to run through a t a n unrestricted rate. The samples are added to the columns 1010
ANALYTICAL CHEMISTRY
1 1 1
y
Found 1.24 1.45 1.90
Std. Dev. 0.028 0.062 0.101
from pipets a t a flow rate of 6 to 12 drops per minute. The aliquot prefepably should contain not more than 6 to 8 y. The fluoride is removed from the resin by stepwise elution with increasing concentrations of sodium acetate, beginning a t 0.1M solution. Ten milliliters of this solution are run into the column at a rate of about 6 to 8 drops per minute, folloived by 10 ml. of 0.2iM, and then 10 ml. of 0.3M solution. The effluent from each treatment is collected in a separate tube. If the sample contains less than 6 to 8 y of fluoride, usually all will appear in the second fraction. If very small amounts of fluoride are expected, collecting the 0.2M effluent in two 5-ml. fractions may enable greater precision of estimation, as with proper adjustment of resin height and rate of flow, the total fluoride may be obtained in 5 ml. of effluent. Each fraction, however, should be checked for fluoride. The 0.3M solution regenerates the resin sufficiently when distillates are analyzed. If the sample contains phosphate, chloride, or sulfate, the resin should be cleaned with 25 ml. of 1M acetate, followed with distilled water to remove excess acetate. T o 5-ml. aliquots of the fractions suspected of containing fluoride are added 0.5 ml. of solution A and 0.5 ml. of solution B. The samples are thoroughly mixed and allowed to stand for 10 minutes or more. The absorbances of the samples are read in a spectrophotometer a t 527.5 m p . The fluoride values are determined from a curve prepared by plotting the absorbance readings of standard fluoride solutions made to volume with 0.2U sodium acetate solution against the concentration of fluoride. One to 5
y per 5 ml. a t intervals of 1 y are convenient standards to use in preparing the curve. It is well to include a few standards with each group of samples to assure that they fall within the expected error limits of the data used in constructing the curve. INTERFERENCES
Willard-Winters distillates are relatively free of interfering ions and with these the resin functions chiefly as a means of concentrating fluoride. Atmospheric and water samples, however, may contain interfering ions. A solution prepared to simulate what might be expected in some water samples contained iron, aluminum, and fluoride, each 0.6 p.pm.; phosphate. 2 p.p.m.; sulfate, 10 p.p.m.; and chloride, 21 P .P*.m. Six replications gave a n average of 0.64 p.p.m. of fluoride, range 0.61 to 0.68 p.p.m. Three replications of the same fluoride solution n-ithout the other ions gave an average of 0.64 p.p.m. of fluoride, range 0.64 to 0.65 p.p.m. The order of emergence of the ions from the column when it is eluted with increasing concentrations of sodium acetate is as follom: Iron and aluminum are not adsorbed and run with the effluent through the column; fluoride, phosphate, chloride, and sulfate are eluted in this order. K h e n 0.2V sodium acetate is used to elute the column, all the fluoride is renioved with 20 ml. of solution. Fiftjto 70 ml. are required to elute the phosphate and 100 ml. fail to remove the chloride and sulfate. These are removed nith 25 nil. of 1N acetate, and this amount is recommended for cleaning columns after they have been used with n ater samples. DISCUSSION A N D RESULTS
Evidence of the precision of determining fluoride in dilute solutions compared with determination after concentration of the ion exchange column is given in Table I. il solution of sodium fluoride containing 0.04 y of fluoride per ml. n as prepared. Fluoride was determined directly on a series of 5-ml, aliquots containing a total of 0.2 y per sample. In another series, 50-ml. aliquots of the same solution ere concentrated by means of the columns to contain 2 y per 5 ml. Animal soft tissues, as a rule, contain rather small amounts of fluoride, usually in the range of 0.02 to 0.2 p.p.m. T o obtain complete recovery of fluoride from the ash of biological samples, 150 to 250 ml. of distillate must be collected. In order to get a distillate in the concentration range that can be measured with acceptable
precision, samples of 50 grams or more are required. The present method furnishes a means of concentrating the fluoride, a t the same time freeing it of ions that would interfere with accuracy of its estimation; thus, precise determinations can be made on much smaller samples. To assess the ability of the method to concentrate small amounts of fluoride in JF7illard-Winter distillates, six 2-7 samples of fluoride were distilled in the usual way, and 100 ml. of distillate were collected. Fluoride was determined directly on a series of 5-ml. aliquots of the distillate. These should contain 0.1 y of fluoride. Of six determinations, the absorbance readings of fire indicated no fluoride was present. Fifty-milliliter portions of these distillates mere then run through the columns, and samples were thus concentrated to 1 y in 5 ml. These showed
*
0.95 0.176 y of fluoride as an average of six determinations. To compare
resin and the fluoride removed and estimated as outlined above.
these values with the original solution from which the distillations were made, a series of 12 determinations was run on aliquots containing 1 y of fluoride per sample. The average result was 0.99 + 0.117 y (Table 11). The manipulative and system errors of the distillations procedure as well as those inherent in the ion exchange and spectrophotometric techniques are included in the determinations of the concentrated distillate. The recovery of fluoride when added to varying amounts of tissue is shown in Table 111. The method has been used in this laboratory for more than a year for determining fluoride in atmospheric samples. Previous distillation of the sample is unnecessary. The sample need only be percolated through the
(1) Attebury, R. W., Boyd, G. E., J. Am. Chem. SOC.72, 4805 (1950). ( 2 ) Bumstead, H. E., Wells, J. C., ANAL.CHEW24, 1595 (1952). (3) Megregian, S., Ibid., 26, 1161 (1954). (4) Milton, R. F., Analyst 74, 54 (1949). (5) Sielsen, J. P., Dangerfield, A. D., A.M.A. Arch. Ind. Health 1 1 , 61 (1955). (6) Powell, W. 8., Saylor, J. H., ANAL. CHEM.25, 960 (1953). ( 7 ) Price, hf. S., Walker, 0. J., Ibid., 24, 1593 11952). (8) Smith, F.-A.,’ Gardner, D. E., Arch. Biochem. 29, 311 (1950). (9) Willard, H. H., Horton, c. A , , ANAL. CHEM.22, 1190 (1950). (10) Willard, H. H., Rinter, 0. B., IND. ENG. CHEM.. ANAL. ED. 5. 7
LITERATURE CITED
RECEIVEDfor review June 18, 1957. Accepted December 16, 1957.
Microdetermination of Silicon in Plants RICHARD J. VOLK’ and ROBERT L. WEINTRAUB Chemical Corps Biological Warfare laboratory, Fort Detrick, Frederick,
A,
spectrophotometric molybdenum blue method has been modified for the determination of microgram quantities of silicon in plant tissue to permit analysis of individual rice leaves. Analyses of tissues containing from 0.1 to 10% silicon are performed conveniently with samples of the order of 25 mg. The silicon is brought into solution b y ashing, fusing the ash with sodium carbonate, and dissolving the melt in acid. Phosphorus, in amounts usually found in plant tissue, does not interfere.
I
AN I ~ V E S T I G A T I O X of the resistance of rice plants to the blast disease, a method was desired for determining the silicon contents of individual rice leaves. As the smallest of these leaves weighed less than 3 mg. and contained as little as 25 y of silicon, a method of high sensitivity was required. The commonly employed gravimetric method for silicon in plant tissue \vas obviously unsuitable, and even the more recent microgravimetric procedures (5) did not appear feasible. ,4 number of colorimetric methods based on the production of yellow molybdisilicic acid or its reduction product, molybdenum blue, have been described, but none was directly applicable to the analysis of plant tissue. The procedure Present address, Department of Soils, North Carolina State College, Raleigh, N . C.
x
Md.
of Mullin and Riley ( 7 ) for the analysis of sea water was selected as the most suitable for modification to analysis of plant tissue. E Q U I P M E N T A N D REAGENTS
Polyethylene ware, rather than glassware, should be employed wherever possible in order to minimize silicon contamination. For some purposesfor example, the storage of liquid samples-paraffin-coated glassware is satisfactory. If neither polyethylene rvare nor paraffin-coated glassivare can be used, as in the cas? of pipets, the glassware must be stored in a nitric-sulfuric acid bath to dehydrate the silica ( 7 ) . Platinum crucibles are used for ashing the plant tissue and for subsequent fusion of the ash with sodium carbonate. A Beckman Model €3 spectrophotometer with 1-em. absorption cells was used. Tl’ater of sufficient purity may be prepared by redistilling water through an all-borosilicate glass still and storing it in polyethylene containers prior to- use. Kater obtained in this manner usually contained less than 6 p.p.b. of silicon. All solutions should be prepared with redistilled water and lowsilicon reagents and stored a t room temperature in wax-coated or polyethylene reagent bottles unless otherwise specified. The properties and limitations of the acidic ammonium molybdate and reducing solutions are discussed by -1Iullin and Riley ( 7 ) . Ammonium molybdate solution. Dissolve 20 grams of ammonium molybdate tetrahydrate in water containing 60 ml.
of 12N hydrochloric acid and dilute to 1 liter. Metol solution. Dissolve 10.0 grams of metol (p-methylaminophenol sulfate) in water containing 6.0 grams of sodium sulfite and dilute to 500 ml. Store a t room temperature in a foilcovered polyethylene bottle. Discard when discolored. Oxalic acid solution, 10% w,/v. Reducing solution. Mix, in a 500-nil. polyethylene bottle, 100 ml. of metol solution, GO ml. of oxalic acid solution, 120 ml. of 9N sulfuric acid, and 20 ml. of water. Store under refrigeration. Allow the reducing solution to attain room temperature prior to use. Sodium silicate stock solution. Prepare a stock solution containing approximately 1.0 mg. of silicon per ml. by fusing pure silica with sodium hydroxide (7). Standard silicate solution. Prepare from the silicate stock solution to contain 5.0 y of silicon per ml. PROCEDURE
Preparation of Plant Sample. Dry tissue samples a t 70’ C. Individual leaf samples weighing less than 50 mg. may be cut up with scissors and the entire leaf used for analysis. Grind larger samples in a Wiley mill t o pass a 20-mesh screen; mix them thoroughly and redry prior to analysis. To avoid silicon contamination during grinding, an aluminum plate may be substituted for the glass d a t e used to cover the blades ofThe mill. Dissolution of Silicon. RaDidlv weigh a 25-mg. or smaller sample i f VOL. 30, NO. 5, M A Y 1958
1011
