V O L U M E 2 5 , NO. 11, N O V E M B E R 1 9 5 3
1741
(3) Kingsley, G.R.,and Schaffert, R. R., Science, 116,359 (1952). Knickmann, E.yon, Z . Pflanrenernahr. Diing. Bodenk., 54, lli (1963). ( 5 ) Kohnlein, J., and Liicke, K. E., E d . , 57, 114 (1952). (6) RIaranis, T.P..llurihead, E. E., Jones, F., and Hill, J. If.,J . Lab. CIiil. M c d . , 32, 1208 (1947). ( i ) Jlosher, R. E., Boyle, A. W., Bird, E. J., Jacobson, S. D., Bachelor, T. Jl., Iseri, L. T., and Myers, G. B., Am. J . Cliii. Prcthol., 19,461 (1949). (8) llosher. R. E., I t a n o , kl., Boyle, A. J., IIwrs, G. B., and Iseri, L. T., Ibid., 21, 75 (1951). (9) Xatelson, S.,I b i d . , 20, 463 (1950).
(IO) Overman, R. R., and Davis, -4.K., J . B i d . Chem., 168, 641
(4)
(194i). (11) Severinghaus, J. W., and Ferrebee, J. W., Ibid., 187,621 (1950). (12) Sniit, J., and tllkemade, C . T. J., Biochim. Biophys. Acta, 6 , 508 (1951). (13) Smith, R. G.,Craig, P., Bird, E. J., Boyle, .I.,Iseri, L. T., Jacobson, S. D., and Myers. G . B.. Am. J . CI1'n. Pathol., 20,263 (1950). (14) Zak, B., Nosher, R. E., and Boyle, A. W , , I b i d . , 23, 61 (1953). RECEIVED f o r review June 8, 1953. Accepted Sugust 21, 1953. Presented in part before the Division of Biological Chemistry a t the 123rd Meeting of the AMERICASCBEMICALSOCIETY, Los Angeles, Calif.
Determination of Fluorides Spectrophotometric ddaptation of Method of Association of Oficial Agricultural Chemists JOSEPH N.ICKEN AND BERNARD M. BLANK Food Research Laboratories, Znc., Long Island City,S. I-. r THE
increasing prevalence of fluoridation of communal water
Color Reagent. Hydrochloric acid, 10 ml. Hydroxylamine hydrochloride, 10 ml. -4lizarin sodium monosulfonate, 40 ml. Thorium nitrate, 40 ml. Color reagent must be freshly prepared. The components are added in the sequence listed. The hydrosylamine was included, as recommended by the AOAC ( 3 ) method, in order to discharge traces of chlorine which might be found in the distilled samples, due to decomposition of the perchloric acid used in the distillation. The amount of hydrochloric acid was chosen so as to give a pH of 2.75 in the final solution. Dahle et al. ( 4 ) found that at this pH small changes do not influence the titer of the thorium nitrate solution-i.e., the ratio of fluorine to thorium-and hence they do not affect the intensity of the color.
1 supplies has brought the determination of fluorine content of
foods to a routine level in some laboratories. A method commonly employed iq that of the Association of Official Agricultural Chemists ( I ) , as reported by Clifford ( S ) , and based on the studies of Dahle et al. (4). This so-called back-titration procedure involves matching the colors of a blank and the sample by a visual method and depends largely on the ability of the analyst to distinguish rolors. The procedure also requires mixing the contents of the tubes several times during the titration, which makes it both time-ronsuming and bothersome. A series of investigations conducted in these laboratories resulted in the development of a spectrophotometric method for the determination of fluorides after prior isolation from food samples by the usual procedures. The proposed method is based on the prinriple of bleaching the thorium-alizarin lake by fluoride ionq.
PROCEDURE
REAGENTS
Stock Solutions. Hydrochloric arid, 0.1 S Hydroxylamine hydrochloride, 1Cr, aqueous solution Alizarin sodium monosulfonate, 0.1yo aqueous solution Thorium nitrate tetrahydrate [Th(NO&.4H*O 1,0.05% aqueous solution Standard sodium fluoride solution containing 10 micrograms of fluoride ion per milliliter. The solution is prepared as follows: The sodium fluoride content of a commercial C.P. sodium fluoride powder is determined by precipitation as calcium fluoride (6); 2.2105 grams of sodium fluoride ( 100ycbasis) are dissolved in distilled water in a I-liter volumetric flask and made to volume. Ten milliliters of this solution are diluted to 1 liter with distilled water to give the final standard solution.
The fluorides are isolated from the food samples by the usual procedure of either single or double distillation, with all the precautions concerning interfering substances such as phosphates, sulfates, and chlorides. The distillation is conducted in a constant temperature still as described by Huckabay, Welch, and Metler (6). The final distillate is carefully neutralized with 0.05 S potassirlm hydroxide using p-nitrophenol as indicator. Aliquots of the distillates containing from 0 to 50 micrograms of fluoride ion are pipetted into 50-ml. volumetric flasks. Standards containing 0, 10, 20, 30, 40, 50, and 500 micrograms of fluoride ion are prepared for each series of assays. Ten milliliters of the color reagent are added to each flask. The flasks are made to volume with distilled water, stoppered, shaken, and allowed to _. stand for 2 hours. The light absorbance at a wave length of 525 mp is determined by means of a Beckman Model DU spectrophotometer equipped with 10-mm. Corex cells, using a slit width of 0.018 mm. The instrument is set for 100% transmittance with the 500-microgram standard. DISCUSSION
*.---.__
-.--_
,400
\,
\ .20°
\.*
\~ \.
\
DO@350
400
150
500
550
WAVE LENGTH, m p
Figure 1. Spectral Absorbance of Thorium-Alizarin Lake - - - - - - - - , Curve a ___ , Curve b - . - ., Curve e
600
The reason for employing the bleached standard (500 micrograms of fluoride) in setting the instrument will be evident from Figure 1. Curve a represents the unbleached lake (standard 0 ) read against distilled water. Curve b corresponds to the bleached standard read against distilled water. Curve c shows the absorbance of the 0 microgram standard as read against the bleached standard. It will be observed that the last curve has a sharp absorption maximum at 525 mp. Figure 2 shows a typical standard curve obtained with the described procedure. Absorbance readings were taken a t 525 mp. This curve has been extended to include points to 100 micrograms per flask. As evidenced by the curve in Figure 2, the
ANALYTICAL CHEMISTRY
1742 Table I.
Effect of Development Period on Light Absorbance Fluoride,
How 0
07 0.586 0.602 0.608 0,609 0.609 0.609 0.608 0.609
1/4
I/¶
1 2 3 4 5
Absorbance a t 525 m r Fluoride, Fluoride,
207 0.538 0.543 0.546 0.548 0.549 0.549 0.550 0.549
407 0.482 0.485 0.487 0.488 0.489 0.490 0.490 0.490
equipped with a 525 mp filter; however, the 520 mp filter was successfully employed. The colorimetric procedure allows accurate estimation of fluoride in concentrations as low as 0.1 microgram per milliliter. APPLICATION OF SPECTROPHOTOMETRIC PROCEDURE TO ANALYSIS O F TEAS
The fluoride contents of a series of commercial samples of blended orange-pekoe tea were determined. Distillates prepared
Table 11. Reproducibility of Spectrophotometric Procedure Fluoride,
y per Flaak
0 10 20 30 40 50
Bbsorbance a t 525 rng
0.640 0.600 0.561 0.529 0.499 0.472
0.639 0.600 0.562 0 529 0.499 0.471
sensitivity decreases slightly for higher concentrations of fluoride ion. Consequently the range from 0 to 50 micrograms of fluoride per flask is recommended. The intensity of the color changes with time. It is evident from Table I that the color becomes stable after 2 hours development for low concentrations of fluorides. However for concentrations of fluoride ions of 40 micrograms or more per flask, a 3hour development period is recommended. Table I1 shows readings obtained on duplicate standards and indicates the high reproducibility of the spectrophotometric method. This procedure allows accurate estimations of fluoride in concentrations as low as 0.05 microgram per milliliter. As mentioned above, the final distillate is neutralized with potassium hydroxide prior to assay. I n order to investigate the effect of potassium ions on the assay the following experiment was undertaken. An amount of potassium (1 ml. of 0.05 N potassium chloride), greater than any that might reasonably be expected to be found in an aliquot taken for analysis, was added to a series of standard solutions of fluoride, and the color reagent was added. No significant change in absorbance was observed compared to untreated standards.
Table 111. Fluoride Content of Orange Pekoe Teas” Tea Sample A
B
C D E 0
Fluoride Content, P.P.M., a8 Determined b y Back-titration Spectrophotometry 111.1 112.7
83.6 91.4 119.2 128.3
85.1 92.9 119.4 126.9
As received.
MODIFICATION O F COLOR REAGENT FOR DETERMINATIONS WITH PHOTOELECTRIC COLORIMETER
In order to permit determinations to be made with an Evelyn photoelectric colorimeter in which tubes of 22.5-mm. diameter are employed, the color reagent was modified. The composition of the modified reagent is as follows: Hydrochloric acid, 0.2 N , 10 ml. Hydroxylamine hydrochloride, 2y0 aqueous, 10 ml. Alizarin sodium monosulfonate, 0.1% aqueous, 40 ml. Thorium nitrate tetrahydrate, 0.05% aqueous, 40 ml. The procedure employed is the same as described above except that 5 ml. of the modified reagent are added to each flask. The range of fluoride concentrations is the same as described for the spectrophotometric procedure. The Evelyn colorimeter is not
.350
.30d 0
20 40 60 80 MICROGRAMS OF FLUORIDE PER 50 ML.
100
Figure 2. Typical Standardization Curve
by the official procedure were read by the back-titration method and the spectrophotometric method described above. The results are shown in Table 111, from which it is seen that excellent agreement was obtained between the two methods. CONCLUSIONS
The modified AOAC method presented allows accurate and reproducible estimations of fluorides in concentrations as low as either 0.05 or 0.1 microgram per milliliter depending on whether a spectrophotometer or photoelectric colorimeter is used. It has the advantage that it employs standard laboratory equipment and does not necessitate the use of 20-mm. or 50-mm. cells as required by other published spectrophotometric procedures (e). LITERATURE CITED
of Official Agricultural Chemists, Washington, D. C., Official Methods of Analysis” 7th ed., 1950. (2) Bumstead, H. E., and Wells, J. C., ANAL.CHEM.,24, 1595
(1) As:ociation
(1952). (3) Clifford, P. A., J . Assoc. O&. Agr. Chemists, 27, 246 (1944). (4) Dahle, D., Bonnar, R. U., and Wichmann, H. J., I b i d . , 21, 459 (1938). (5) Huckabay, W. B.. Welch, E. T., and Metler, A. V., IND.ENG. CHEM.,ANAL.ED., 19, I54 (1947). (6) Scott, W. W., “Standard Methods of Chemical Analysis,” Vol. I, 4th ed., p. 226, New York, D. Van Nostrand Co., 1925.
RECEIVED for review March 12, 1953. Accepted June 24, 1953.
