‘A’” Figure 7. Idealized melting curve for constant thermal head
the slopes of T. before melting has started and after it has finished will be re = hk/(nC,
+ C,)
ri = hk/nCi
+ C,)
and
respectively, where subscripts c and The heavy dashed lines represent extrapolation of these slopes t o To. It may be readily shown that
1 refer t o crystals and liquid.
Z
=
h(tz
AH
=
kh(ts
Y
=
- ti) - ti)/n h(t” - t-’)
F-1
=
(t,
-
tl)/(t”
- L’)
Thus no measurements of area are necessary. If the apparatus were to be operated in this manner, the block would be made lighter for quicker response; and if measurements were made below room temperature, it would be necessary to provide cooling as well as heating. It seems doubtful that the added complicatiocs would give a n y great improvement in results. The temperature range of the apparatus is limited at the lower end t o melting points ca. 20” above the boiling point of nitrogen and at the upper end to around 150”,imposed by tape on the sample thermocouple support, grease in the block thermocouple well, and the enamel insulation. Rather minor modifications wroula, however, permit operation well u p toward the softening point of borosilicate glass. Although the present apparatus has been used with samples of ea. 0.3 ml., i t could doubtless be used down t o 0.1 ml. or somewhat lower Kith smaller sample bulbs without great loss in sensitivity and accuracy. Evaluations of the slope of the F-’ us. T plot and of Z1 for highly impure samples are somewhat subjective. The present measurements on solutions of known contamination were not blind and hence are subject to observer bias. However, it is believed that different
operators should usually reproduce the evaluation of N? to between 10 2nd 20%. LITERATURE CITED
( 1 ) -4nal. Chim. d c t a 17. 1-174 (19571 - , ( 2 ) Ginnings, D. C., Furukawa, G . T., J . A m . Chem. SOC.75,522 (1953). (3) Mathieu, M. P., Bull. SOC. C‘hzm. Belges 63, 333 (1954). (4) Oliver, G. D., Eaton, M.,Huffnisn, H. IM., J . Am. Chem. Soc. 70, 1502 (1948). (5) Rossini, F. D., Wagman, I). D., I -
\ - - - - ,
Evans, W. H., Levine, S., Jaffe, I., Natl. Bur. Standards (U. S.),Circ. 500
(1952). (6) Shenker, H., Lauritzen, J. I., Jr., Corrucini, R. J., Lonberger, 8.T., Zbid., 561 (1955). ( 7 ) Skau, E. L., Proc. A m . Acad. .4rts Sei. 67, 551 (1932); J. Phys. C‘hem. 37, 609 (1933). (8) Skau, E. L., Arthur, J. C., Jr., Wakeham, H., “Determination of Mel!ing and Freezing Temperatures,” in “Technique of Organic Chemistry,” Vol. 1, “Physical Methods,” 3rd ed., A. Weissberger, ed., p. 287, Interscience, New York, 1959. (9) Sturtevant, J. M., “Calorimetry,” Ibid., 1, p. 523. (10) Timmermans, J., “The Physico-
Chemical Constants of Binary Systems in Concentrated Solutions,” Vol. 4, p. 481, Int,erscience, New York, 1960. RECEIVEDfor review March 20, 1962. Accepted June 11, 1962. Work done under the auspices of the U. S. Atomic Energy Commission.
Isolation and Determination of Microgram Amounts of Fluoride in Materials Containing Calcium and Orthophosphate H. WHITNEY WHARTON Research and Development Department, Miami Valley laboratories, Procter & Gamble Co., Cincinnati, Ohio
b Up to 4 pg. of fluoride can b e separated from dental enamel by gaseous diffusion of HF from an HClOd solution of the sample in a polypropylene Conway diffusion dish. The HF is trapped in NaOH. The resulting fluoride i s determined spectrophotometrically by an improved Zr-SPADNS bleaching method. The accuracy and reproducibility of the entire method are to k0.05 pg. of fluoride a t the 1-pg. level, k0.07 pg. at the 3-pg. level. The spectrophotometric method has been applied successfully to the condensates of Willard and Winter type distillations.
H
in a recent review ( 5 ) , summarized the extensive work t h a t has been done on the isolation a n d determination of fluoride. Spectrophotometric methods are most popular ORTON,
1296
0
ANALYTICAL CHEMISTRY
for determining amounts of fluoride under 10 pg. Polarographic and amperometric methods have also been proposed. Spectrophotometric methods for fluoride generally utilize colored metal-dye complexes or lakes. Fluoride ion bleaches the color by removal of the metal ion from the metal-dye complex. The color bleaching is usually proportional t o the amount of fluoride present. The method of Belcher, Leonard, and West ( 1 ) involves positive color development rather than bleaching. This method, improved by Greenhalgh and Riley (S), is indicated as applicable t o the range of 10 to 50 fig. of fluoride. On the basis of a literature survey of the characteristics of various fluoride methods, the spectrophotometric method of Bellsck and Schouboe (9 was selected as being superior for our purposes. It appeared most suitable for routine
use, as the required reagent consists of a single phase, is easily prepared, and remains stable indefinitely. Its response to fluoride conforms to Beer’s law and i t is at least as sensitive as most other methods. Since the reagent is well buffered at a p H of less than 1, relatively large changes in acid concentration can be tolerated. None of the other literature methods appear t o possess all of these desirable characteristics. I n the original work of Bellack and Schouboe the reagent consisted of a n HC1 solution containing zirconium and SPADNS [4,5-dihydroxy-3-(p-sulfophenylazo) - 2,7- naphthalenedisulfonic acid, trisodium salt] in a 1 to 4 molar ratio. It was found in the present study that the spectrophotometric sensitivity is markedly increased by decreasing the Zr-SPADNS ratio t o 1 to 12. This modification is described below.
The Zr-SPADNS method requires that fluoride be free of certain interferences, among them calcium and phosphate ions. The determination of fluoride in dental enamel requires the complete separation of the fluoride from a sample matrix consisting principally of calcium hydroxyapatite, Calo(OH)r (pod)? Various methods have been proposed for the isolation of small amounts of fluoride. Micro amounts of fluoride have been isolated by an excellent but relatively complicated microdistillation method (11). Other variations of the classic Willard and Winter distillation technique have been published, but these require upwards of 100 ml. of distillate. This makes the recovery an accurate determination of 0.5 fig. of fluoride unfeasible. Ion exchange techniques have been used (IS), but dilution effects and introduction of foreign salts can interfere. Diffusion techniques, using polyethylene bottles, have been applied t o larger amounts of fluoride with good success (4,12). A diffusion technique using simple polypropylene Conway dishes was developed in this study.
Figure 1. Schematic diagram of microdiffusion cell showing relative locations of materials for F- diffusion
various concentrations of N a F which have been carried through the diffusion operation. Diffusion recovery checks can be made by adding appropriate amounts of NaOH and wetting agent to undiffused N a F standards. The previously described, improved Zr-SPADNS method may, of course, be applied to Willard and Winter distillates. Proper spectrophotometric conditions and Zr-SPADNS requirements for various concentrations of F- are given in Table I. The spectrophotometric method is unaffected by any HClO, that may be carried over in the distillate.
was from Nuclear Products Co., El Monte, Calif. Procedure. Wipe the dry diffusion cells with the static-removing brush RESULTS - DISCUSSION and weigh the sample containing from 0.3 to 2.5 pg. of fluoride directly into Spectrophotometric Method. The the inner annular chamber of the cell Zr-SPADNS method of Bellack and (see Figure 1). Unless removed, the Schouboe uses a 1 to 4 molar ratio of static charge is sufficient to expel some Zr to SPADNS, following the lead of of the sample and, more seriously, to Megregian (7), who determined that introduce sample into the inner comthis ratio of metal to dye was the most partment. Place 0.3 ml. of 1.3N NaOH in the center chamber. Place 3 drops sensitive for fluoride using a Zr-Erioof diluted wetting agent and 0.3 ml. chrome Cyanine R lake. Other workers of water in the inner annular chamber. (6) have used as low as a 1 to 8 metal to (With samples containing appreciable dye molar ratio to improve sensitivity. amounts of COS-* such as teeth or The 1 to 4 molar ratio of Zr to SPADNS bone, place the wetting agent and water gives a spectrophotometric sensitivity of directly on the solid samples to slow 0.0031 pg. of F per sq. cm. as defined down the attack of the acid and reduce EXPERIMENTAL by Sandell (10). Decreasing the metalspattering.) Place 0.5 ml. of condye rati; to 1 to 8 and to 1 to 12 gives centrated perchloric acid in this same Reagents and Apparatus. REAGENT chamber, but away from the sample. sensitivities of 0.0020 and 0.0015 pg. of A. S P A D N S [4,5-dihydroxy-3-(pLiberally grease the edge of the clear F per sq. cm., respectively. The latter sulfophenylazo) - 2,7 - naphthaleneplastic top with a silicone-type grease corresponds to a molar absorptivity of disulfonic acid, trisodium salt], Eastand place over the diffusion cell. 12,700. The 1 to 16 molar ratio is only man Organic Chemicals, No. 7309 Tilt and revolve the sealed cells to slightly more sensitive to fluoride than (3.16 grams) is dissolved in 550 ml. of mix the contents of the annular chamber the 1 to 12 ratio. The 1 to 12 molar deionized water. thoroughly, place in the heated desicratio of Zr to SPADNS, selected for REAGENT B. Zirconyl chloride octacator, and maintain a t 60' C. for 24 use, gives an absorbance of about 0.5 for hydrate (0.133 gram) is dissolved in 50 hours. Heating causes the NaOH to ml. of deionized water. Concentrated 1.0 ml. of Zr-SPADNS solution (equal wet the cell and spread out uniformly, HCl (350 ml.) is added and the resulting aiding the capture of diffused fluoride. volumes of reagents A and B) in 10-ml. solution is diluted to 500 ml. with deAfter this period, cool to room temtotal volume using 1-cm. cells in the ionized water. perature. Fill the inner chamber, conBeckman Model B set a t sensitivity 2 REFERENCESOLUTION. Fifty millitaining the NaOH and the resulting using an 0.1-mm. slit. liters of reagent A is added to 500 ml. NaF, with deionized water. Transfer The possible existence of Zr-SPADNS of deionized water and 35 ml. of conthe contents of this chamber by Pasteur complexes was examined by application centrated HC1 is added. This solution pipet to volumetric flasks of approof the mole ratio method. A continuis used to set the zero absorbance (100% priate size. Repeat this washing operaous increase in net absorbance as the 2') of the spectrophotometer. It is tion four times. For 0 to 4 pg. of stable and re-usable. metal-dye ratio was varied between 1 to fluoride, add 1.0 ml. of the Zr-SPADNS SINGLESPECTROPHOTOMETRIC REAsingle spectrophotometric reagent and 1 and 1 to 16 was obtained for systems GENT. Equal volumes of reagents A dilute the solutions to 10.0-ml. total in which Zr concentration was held conand B are mixed. As the mixture is volume. Read the absorption in 1-cm. stant and SPADNS concentration varsteble indefinitely, it is convenient to cells a t 590 mp (or observed A,), with ied, or for the reverse situation. The combine those solutions remaining after the reference solution in the reference increase in absorbance tapered off to a preparation of the reference solution. cell. nearly constant value beyond the 1 to 16 STANDARD FLUORIDE SOLUTION.Dry, Zr-SPADNS ratio. The above data A standard curve is constructed using reagent grade N a F (22.1 mg.), dissolved in 1 liter of deionized water, provides a solution containing 10.0 pg. of Fper ml. As diffusion standards, microTable 1. Spectrophotometric Conditions and Zr-SPADNS Requirements for Various liter aliquots may be used without inConcentrations of Fcreasing the sample solution volume Total significantly. Volume, Cells, The polypropylene microdiffusion F-, rg. MI. Zr-SPADNS, M1. Reference Soln. Cm . cells, size 44, are available from the As prepared. I 0-4 10 1 . 0 Aloe Scientific Co. The wetting agent, 1.0 6 drops Dil. 1: 1 with H10 5 0-4 25 Tergitol, accompanies the diffusion cells concd. HClOd and is diluted to 0.1% with deionized As prepd. 1 0-8 25 2.0 water. The heated desiccator was 2.0 6 drops Dil. 1: 1 with HZO 5 0-8 50 concd. HClOd Harshaw No. H-18880; the Pasteur AE prepd. 1 5-40 100 10.0 pipets were Harshaw No. H-55698; the Staticmaster brush, No. 1S200,
+ +
VOL 34, NO. 10, SEPTEMBER 1962
1297
Table 11. Recoveries of F- Added as NaF from a Variety of Matrices, by Microdiffusion
Sample Matrix NaF only
F-, pg. Added Founda
NasP040 Ca(NO&C Caa(Pn4)zd
0.50 1.00 1.50 2.00 2.50 3.00 3.50 1.40 2.80 1.40 2.80 0.50 1 00
0.49 1.03 1.50 1.98 2.43 2.89 3.35 1.39 2.75 1.43 2.81 0.48 1.02
2.00 3.00
2.02 2.82
i.50
i.50
Recovery,
%
98 103 100 99 97 97 96 99 98 102 100 96 102 100 101 94
Over-all reproducibility, f O .05 pg. F- at 1-pg. level, f 0 . 0 7 pg. F- at 3-pg. level. * PO4-* equivalent to 10 mg. Ca3(PO&. Ca+* equivalent to 10 mg. Cas(POa)r. d Corrected for 21 p.p.m. F- found in a
caU(po4)z.
suggest that no distinct Zr-SPADNS complexes exist over the mole ratios examined. Application of the method of Job is questionable at the high ratios of dye t o metal expected for this system. It is interesting t h a t a 1 t o 2 ZrSPADNS complex has been reported in 5N HClOi (8). The maximum color intensity of the 1 to 12 Zr-SPADNS reagent is not obtained until about 3 weeks after preparation. However, reaction of the aged (or unaged) reagent with fluoride is instantaneous. The aged Zr-SPADNS reagent is stable with regard t o absorbance values and sensitivity to fluoride for at least 2 weeks in daylight; in the presence of fluoride the solutions are stable for at least 3 weeks in daylight. After 6 months’ storage of the ZrSPADNS reagent in brown bottles there was no significant change in net absorbance of the reagent and only a 3% increase in sensitivity toward fluoride.
Table 111.
Sample
The wavelength of maximum absorbance for all metal-dye ratios ranged from 590 to 595 mp for two lots of dye as determined on Cary Model 14 and Beckman Models B and D U spectrophotometers. The wavelength of niaximuni absorbance of .570 mp reported by Bellack and Schouboe appears on the side of the rapidly rising absorbance due to the dye itself. This may account for some of the difficulties encountered in evaluating this method (9). The Zr-SPADSS reagent (1 to 12 ratio) exhibits linear bleaching by fluoride over the 0 t o 4 pg. range. Above 4 pg. of fluoride, bleaching per unit of fluoride decreases. The ratio of 4 pg. of fluoride per milliliter of ZrSPADNS reagent corresponds to a Zr/F ratio of 2 to 1. Microdiffusion and Spectrophotometric Determination of Fluoride. T h e recovery of fluoride diffused from N a F solutions and from solid samples of Ca(N0J2, Na3P04, and Ca3(P04)2 containing added N a F is indicated in Table XI. The recoveries from N a F solutions were substantiated by analyzing the HC104 solution directly for residual fluoride by the Zr-SPADNS method. The time necessary for such recoveries could be reduced to about 8 hours by agitating the samples. For routine analysis of large numbers of samples, the longer and more reliable static diffusion offers no disadvantages. By triple decking the desiccator plates, 21 samples can be diffused simultaneously. Analyst time for batch’runs is about 20 minutes per sample. The fluoride content of typical dental enamel samples as determined by niicrodiffusion and Zr-SPADNS is shown in Table 111. Samples A and B were analyzed by both the original Bellack and Schouboe 1 to 4 Zr-SPADNS and the 1 t o 12 Zr-SPADNS systems. The standards were N a F solutions carried through the entire procedure. For low total fluoride, the 1 to 12 Zr-SPADNS system markedly decreases the standard deviation; for larger amounts, a more precise analysis is also obtained. Sample B was analyzed by independent
Typical Results for Fluoride Content of Dental Enamel Samples as Determined by Microdiffusion and Zr-SPADNS 1 : 12 Zr-SPADNS 1 :4 Zr-SPADNS ( 2 )
P.p.m. F-
u
Av. pg. F-detd.
No. of
Detns.
P.p.m. F-
u
Av. pg. F-detd.
NO. of
Detns.
a Independent value of 437 p.p.m. F- obtained following Willard and Winter distillation and Zr-SPADNS spectrophotometry.
1298
ANALYTICAL CHEMISTRY
workers using a Willard and Winter steam distillation and the Zr-SPADNS spectrophotometric method and contained 437 p.p.m. of F-. This compares favorably with the value of 442 p.p.m. of F- found after microdiffusion. The standard deviations are +0.05 pg. of F- at the 1-pg. level and d=0.07 k g . of F- a t the 3-ug. level, using the 1 to 12 Zr-SPADNS following isolation of the fluoride by microdiffusion. While this microdiffusion method was developed specifically to separate fluoride from the matrix of dental enamel, it has been applied successfully to samples of bone, toothpaste, agar-agar, and other sample matrices containing ionic fluoride. CONCLUSION
The major advance provided by this method for fluoride determination is that fluoride can be accurately determined in samples such as teeth and bones which contain large amounts of calcium and phosphate and low levels of fluoride, and which are in short supply. The spectrophotometric method is simple, sensitive, and fast. By the technique of microdiflusion, the isolation, concentration, and quantitative recovery of up to 4 pg. of inorganic fluoride are accomplished using simple, inexpensive equipment and a minimum of manual operations. LITERATURE CITED
(1) Belcher, R., Leonard, M. A,, West, T. S., J. Chem. SOC.1959,3577. (2) Bellack, E., Schouboe, P. J., ANAL. CHEM.30, 2032 (1958). (3) Greenhalgh, R., Riley, J. P., Ana[. Chim. Acta 25,179 (1961). (4) Hall, R. J., Analyst 85, 560 (1960). (5) Horton, C. A,, in “Advances in An-
alytical Chemistry and Instrumentation,” Vol. 1 , C. N. Reilley, ed., pp. 151-99, Interscience, New York, 1960. (6) Jankovic, S., Croat. Chem. Acta 32,
165 (1960). (7) Megregan, S., ANAL. CHEM. 26, 1161 (1954). (8) Peshkova, V. M., Mel’chakova, N. V., Sinitsyna, E. D., Izv. Vysshikh Ucheb. Zavedenii Khim. i Khim. Tekhnol. 3, 72 (1960); Anal. Abstr. 8, 3659.(1961). (9) Robert A. Taft Sanitary Engineering Center, U. S. Dept. of Health, Educa-
tion, and Welfare, Public Health Service, Analytical Reference Service, Sample Type 111-B, Water, Fluoride, Cincinnati, Ohio, August 1961. (10) Sandell, E. B., “Colorimetric D;: terminations of Traces of Metals, 2nd ed., pp. 45-50, Interscience, New York, 1950. .) Singer, L., Armstrong, W. D., ANAL
HEM.
31, 105 (1959). (12) Stegemann, H., Jung, G. F., Z. physiol. Chem. 315, 222 (1959). (13) Talvities, N. A., Brewer, L. A., A m . Ind. H y g . Assoc. J . 21, 287 (1960).
RECEIVEDfor review February 28, 1962. Accepted June 18, 1962. Division of Analytical Chemistry, 14156 Meeting, ACS, Washington, D. C., March 1962.
