tions containing ammonium, thallous, and a variety of other ions. As Table I11 indicates, interference due to ammonium can be eliminated by adjusting the pH to about 12. Silver interferes seriously and this ion should be removed. Iron(III), zinc(II), copper(II), and cadmium(I1) do not interfere when the titration i s carried out in 0.2F EDTA at a pH of 8. However, EDTA does not eliminate the interference due to thallous ion. This species must be removed by precipitation, by ion exchange, or by solvent extraction. The mercurization of tetraphenylborate according to reaction 3 is well known and, consequently, mercury will interfere in the titration. EDTA does not prevent the reaction (5). Quarternary ammonium salts are quantitatively precipitated
by the titrant and therefore will be a serious interference unless removed from the solution. Protonated amines react with tetraphenylborate ; however, in basic solution no interference is anticipated (5).
The immunity of the titration to high salt concentration is illustrated by the results in 0.2F EDTA which contains sodium at a concentration of 0.40F, i.e., in 1000% excess over the potassium concentration. The thermometric titration of potassium can be carried out in the presence of excess salt or base, but not mineral acid, with no concomitant decrease in precision. One would under such circumstances prefer thermometric titration to either the conductometric or ionselective potentiometric titration. The titration can be used to analyze potassium, ammonium, or thallous solutions with precision and accuracy of better than 1 in the optimum concentration range of 12 to 30mM. In order to further reduce the sample size, one would have to develop a stirring technique which generates less mechanical heating and electronic noise. If larger amounts of sample are available in a matrix free of interfering ions, the conductometric or potentiometric titration procedures should be preferred, since where applicable they are generally more precise and accurate. RECEIVED for review November 4, 1970. Accepted January 25,1971.
A New Approach to the Microdetermination of Fluoride Adsorption-Diff usion Technique P. Venkateswarlul and P. Sita2 Departments of Biochemistry, Sri Venkateswara Medical College, Tirupati, and Postgraduate Institute of Medical Education and Research, Chandigarh, India
THE CONDITIONS for quantitative adsorption of fluoride on magnesium oxide and the application of the findings to the determination of fluoride in water and some biological materials, by procedures involving distillation but not requiring evaporation or ashing, were reported by Venkateswarlu and Narayanarao (1-5). At about this time Singer and Armstrong (6), for the first time, introduced the diffusion technique for isolation of fluoride. Attempts at combining these two procedures to develop an adsorption-diffusion technique, which does not require ashing, for the determination of fluoride in biological materials were unsuccessful. The fluoride adsorbed on magnesium oxide was not available for quantitative diffusion under the experimental conditions of the trials (7). However, if the diffusion technique is applicable to the analysis Present address: Department of Biochemistry, Medical School, University of Minnesota, Minneapolis, Minn. 55455 Present address: Department of Laboratory Medicine, University of Minnesota Hospitals, University of Minnesota, Minneapolis, Minn. (1) P. Venkateswarlu and D. Narayanarao, Indian J. Med. Res., 41,
253 (1953). (2) Ibid., 42, 135 (1954). (3) P. Venkateswarlu and D. Narayanarao, ANAL.CHEM.,26, 766 (1954). (4) P. Venkateswarlu and D. Narayanarao, Indian J. Med. Res., 45, 273 (1957). (5) Ibid., p 369. (6) L.Singer and W. D. Armstrong, ANAL.CHEM., 26,904 (1954). (7) L.Singer, W.D. Armstrong, and P. Venkateswarlu,unpublished data (1955). 758
ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
of fluoride in bone and tooth minerals (6), which are essentially “calcium phosphate” in nature, an adsorption-diffusion technique for the determination of fluoride in biological materials seems feasible, if only fluoride in such samples could be quantitatively adsorbed on calcium phosphate. With these objectives in view, some of the factors governing the adsorption of fluoride on calcium phosphate were now investigated along the lines of which the adsorption of fluoride on magnesium oxide was studied earlier (2). On the basis of the observations, procedures were devised for the quantitative adsorption on calcium phosphate of fluoride present in natural waters, salt solutions, urine, and plant extracts. Calcium phosphate with the adsorbed fluoride was then sedimented by centrifugation and subjected to the microdiffusion technique. The fluoride in the diffusate was analyzed by a suitable spectrophotometric procedure. For analysis of fluoride in the microgram and submicrogram range, it is necessary to keep the overall experimental blank as low as possible. Although the diffusion apparatus blank is very low, calcium phosphate, which is prescribed in this procedure for adsorption of fluoride, could provide appreciable fluoride blank. Several analytical reagent grade samples of calcium phosphate have been found to contain 20 to over 100 pg F/g. A procedure is described in this paper for obtaining calcium phosphate with very low content of fluoride, less than 0.5 ppm, starting from specially purified calcium acetate and potassium dihydrogenphosphate (8). (8) P. Venkateswarlu, W. D. Armstrong, and L. Singer, Indian J . Med. Res., 54, 1156 (1966).
EXPERIMENTAL. Reagents. FLUORIDE-LOW CALCIUM PHOSPHATE. Calcium acetate is purified with three successive half-hour adsorptions by boiling with light magnesium oxide (20 g MgO per 3 liters of 1.5N salt solution) and potassium dihydrogenphosphate and ammonium chloride are purified by three recrystallizations (8). One liter of 0.12M potassium dihydrogenphosphate and one liter of 0.2M calcium acetate solution are added slowly in equal volumes to three liters of boiling solution of 0.001M ammonium chloride and the pH is maintained just alkaline to phenolphthalein by addition of concentrated ammonia. The final reaction mixture is boiled for 15 minutes and cooled. The calcium phosphate is centrifuged down and repeatedly washed with plenty of doubly distilled water until the washings are free from chloride, dried at 110 "C,and pulverized to a fine powder in a clean agate mortar. Accurately weighed amounts of calcium phosphate powder are employed for adsorption of fluoride from the various samples. FLUORIDE-LOW MAGNESIUM NITRATE,0.2M. Magnesium nitrate is also purified by the magnesium oxide adsorption technique. (A) Preliminary Studies on Adsorption of Fluoride Ion on Calcium Phosphate. Twenty ml of solution containing 100 pg fluoride was stirred continuously with 20 mg of fluoridelow calcium phosphate for periods ranging from 5 minutes to 1 hour and at temperatures varying from 25 to 100 OC. At the end of the adsorption period, the calcium phosphate with the adsorbed fluoride was immediately separated from the rest of the solution by rapid filtration and washed twice with 10 ml of doubly distilled water. The adsorbed fluoride was isolated by the diffusion technique of Hall (9), which is a modification of the original technique developed by Singer and Armstrong (6). The diffused fluoride which was trapped by 10 p1 of 0.4M magnesium nitrate solution, in place of magnesium succinate used by Hall ( I O ) , was analyzed by the modified aluminum-eriochromecyanin R method ( I I ) . The same amount of magnesium nitrate as used for trapping the fluoride was also incorporated in the standard fluoride solutions. (B) Adsorption-Diffusion Technique for Determination of Fluoride in Selected Materials. (i) WATER. A suitable aliquot of the water, 5-10 ml, is boiled for 5 minutes with 20 mg of fluoride-low calcium phosphate, cooled to room temperature, and centrifuged for 15 minutes at 3000 rpm. The fluoride adsorbed on to calcium phosphate is determined as in (A), described above. (ii) SALTS. Solution of 0.1-1.0 g of the salt in 20 ml doubly distilled water is boiled for 5 min with 20 mg fluoridelow calcium phosphate. The fluoride adsorbed on calcium phosphate is determined as in (A), described above. (iii) URINE. All urine samples are checked for pH and carefully examined for any sediment. Sediment, if found, is brought into solution with the minimum amount of dilute hydrochloric acid and the pH is readjusted to 6. A suitable aliquot of urine is diluted to 20 ml with doubly distilled water and the fluoride is adsorbed on to calcium phosphate and determined as in (A), described above. (iv) PLANTEXTRACTS.Extracts of plant material (tea) are also processed and analyzed as in (B, i), described above. (v) SERUM. Twenty milligrams of fluoride-low calcium phosphate are added to 5 to 10 ml of serum in a platinum dish and the pH is adjusted so that it is alkaline to phenolphthalein with 5N sodium hydroxide. The contents are evaporated down with frequent stirring, dried at 110 "C, and ashed overnight at 500 O C . The ash is dissolved in 2 ml of 0.5N perchloric acid and transferred to a borosilicate glass test tube. (9) R. J. Hall, Analysr, 85, 560 (1960). (10) Ibid.,88, 76 (1963). (1 1) P. Venkateswarlu, W. D. Armstrong, and L. Singer, Indian J . Med. Res., 54,455 (1966).
6Ooc
4Ooc
25Oc
0
$
1 IO
20
30
ADSORPTION
40
50
60
TIME I N MINUTES
Figure 1. Effect of time and temperature on adsorption of fluoride (100 pg in 20 ml) on calcium phosphate (20 mg) The solution is neutralized with 0.5N ammonium hydroxide to precipitate calcium phosphate and boiled for 5 minutes. The fluoride adsorbed on calcium phosphate is determined as in (A), described above.
RESULTS AND DISCUSSION From preliminary work, it was found that the maximum amount of fluoride that could be adsorbed on the sample of calcium phosphate employed in this study was 10 pg/mg calcium phosphate. In the experiments designed to study the effects of time and temperature on the adsorption of fluoride on calcium phosphate, the initial amount of fluoride in the solution was adjusted to equal 50% of the maximum fluoride-adsorbing capacity of the amount of calcium phosphate employed. The amount of fluoride adsorbed on calcium phosphate increased with time and temperature. One hundred per cent adsorption of fluoride on calcium phosphate was achieved when 20 ml of solution containing 100 pg fluoride was boiled with 20 mg calcium phosphate for 5 min (Figure 1). Five-minute adsorption at 100 "C was therefore followed in the procedures described above for the determination of fluoride by the adsorption-diffusion technique. The marked effect of temperature on the adsorption of fluoride on calcium phosphate observed in the present investigations was not noticed by McCann (12) in his studies on the reaction of fluoride ion with several calcium phosphate preparations. In the present study, the adsorption periods were limited to 5 to 60 min and maintained identically when the same amounts of calcium phosphate were initially exposed to identical amounts and concentrations of fluoride for study of adsorption at different temperatures. In the studies by McCann, the adsorptions were carried out for different periods of time for different temperatures and were also extremely pro(12) H. G. McCann, J . B i d . Chem., 201, 247 (1953). ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
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Table I. Determination of Fluoride by Calcium Phosphate Adsorption-Diffusion Technique Fluoride ppm, Recovery,“ Material Sample No. and Size Mean f Std Dev (N) Mean f Std Dev (N) Upland surface water A 20 ml 0.08 i= 0.014 (4) 98.7 f 3.2 (4) Bore-well water A 5ml 0.30 f 0.024 (6) 98.2 f 4.8 (3) Sodium chloride A 1OOOmg 0.70 f 0.08 (7) 99.4 =t3.6 (6) “AnalaR” B.D.H. Trisodium phosphate BDH A 50mg 36.40 i= 2.4 (4) 96.8 f 4.2(6) Calcium nitrate BDH A 50mg 57.80 f 3.3 (4) 98.7 & 3.4(6) A 100mg 14.00 =t0.59 (8) Common salt (table salt) B 100mg 17.70 f 1.04 ( 5 ) 98.3 f 3.7 (6) C 100mg 20.00 f 1.40 (4) Urine A 5ml 0.52 f 0.02 (4) B 5ml 0.66 i= 0.02 (4) c 2nd 0.79 f 0.08 (4) 100.3 i:4.8 (18) D 2ml 0.88 f 0.07 (6) E 2ml 1.86 f 0.04 (4) Blood serum (goat) A 10ml 0.13 f 0.01 (15) 99.5 & 5 . 5 (14) Tea A 50mg 54.00 & 2.3 (4)b 99.4 5 7.2(11) B 50mg 60.00 f 1.3(5)* a 2 pg F added to the samples prior to following the prescribed procedures. b Hot water extractable fluoride. tracted for periods of “time varying for several days for looo samples to 1 to 4 months for the lower temperature samples.” These circumstances seemed to have masked the effect of temperature on adsorption of fluoride by calcium phosphate which was evident under the experimental conditions of the present study. Another possible point of difference in the two studies is the discrepancy in the initial fluoride contents of the samples of the calcium phosphate employed in the two studies. In the present study, specially prepared fluoride-low calcium phosphate (0.4 ppm F) was employed. The initial fluoride contents of the samples of calcium phosphate employed by McCann have not been mentioned. Some batches of analytical reagent grade calcium and phosphate salts are known to contain appreciable traces of fluoride (See also Table I). Table I contains the results of fluoride analysis of water samples and a few selected salts, urine, tea extract, and serum, employing the calcium phosphate adsorption-diffusion technique. Except in the case of serum, which was ashed, fluoride was adsorbed from unashed samples. Recovery of 2 pg F added to all samples was satisfactory. Adsorption of fluoride present in serum is not possible at 100 “C because of the problem of heat coagulation of serum proteins. Fluoride present in or added to salt solutions could be adsorbed at room temperature on calcium phosphate precipitated in situ, on addition of calcium nitrate and potassium dihydrogen phosphate solutions to the sample at neutral pH. Attempts at in situ precipitation of calcium phosphate in the presence of serum were unsuccessful, presumably because of the protective action of serum proteins on colloidal calcium phosphate that might have been formed. Nevertheless, the calcium phosphate adsorption technique was applied to the solution of serum ash to get rid of the chloride. This step would eliminate the need for the use of silver-perchlorate to withhold chloride from diffusion. Alternatively, an adequate amount of sodium hydroxide might be used to trap the chloride. This would require incorporation of an equivalent amount of sodium chloride in the standards, compromising to some extent the sensitivity of the spectrophotometric method. Determination of fluoride contamination in nitrates by the straight diffusion or distillation techniques, in conjunction with a colorimetric procedure, would not be very practical because of the nitrous fumes evolved, which exert an oxidizing effect on the dyes employed in the colorimetric procedures. The problem of analysis of bromides and iodides by straight 760
ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
diffusion or distillation techniques is also complicated by the liberation of HBr and HI, which readily dissociate. Such salts can be conveniently handled by the adsorption-diffusion technique described here. Results of analyses of fluoride present in calcium nitrate, employing the calcium phosphate adsorption-diffusion technique, are included in Table I. When 25 perchloric acid extracts of some plant materials were diffused without ashing according to the diffusion technique proposed for animal tissues (13) and analyzed by two different colorimetric procedures ( / I , 14), false high fluoride values were obtained (15). Similar experiences have been reported in the case of fluoride analysis of plasma (16, 17). The advantage of the calcium phosphate adsorption-diffusion technique is that the supernatant, which contains the large bulk of interfering substances, is discarded. Furthermore, it is possible to concentrate traces of fluoride from a large amount of a sample low in fluoride, a feature which permits more reliable determination of fluoride. Except in the case of serum, the present procedure precludes the need for ashing of samples to get rid of interfering substances and also averts the danger of loss of fluoride due to volatilization or contamination with extraneous fluoride during ashing. Furthermore, the fluoride adsorbed on calcium phosphate can be determined directly with the fluoride ion electrode after dissolving the calcium phosphate in a suitable buffer (18, 19), and the diffusion step can be altogether omitted. However, results presented in this report were not obtained with the fluoride electrode. Whether the fluoride adsorbed on calcium phosphate from biological materials, which are often complex in their composition, represents total fluoride or a particular fraction thereof is not answered in this report and is under investigation. RECEIVED for review August 21, 1970. Accepted December 7,1970. This work was supported by a grant from the Indian Council of Medical Research, New Delhi, India. (13) L. Singer and W. D. Armstrong, Anal. Biochem., 10, 495 (1965). (14) L. Singer and W. D. Armstrong, ANAL.CHEM., 31,105 (1959). (15) K. Raman and P. Venkateswarlu, unpublished data (1966). (16) D. R. Taves, Nature, 211, 192 (1966). (17) F. H. Cox and 0. B. Dirks, Curies Res., 2,69 (1968). (18) H. G. McCann, Arch. OrulBiol., 13,475 (1968). 40,613 (1968). (19) L. Singer and W. D. Armstrong, ANAL.CHEM.,
