Determination of Fluoride in Biological Samples by a Nonfusion Distillation and Ion-Selective Membrane Electrode Method Paul J. Ke, Lloyd W. Regier, and Henry E. Power Fisheries Research Board of Canada, Halifax Laboratory, Halifax, Noca Scotia A nonfusion distillation technique has been developed to prepare biological samples for a rapid determination of fluoride content using a solid-state membrane electrode. The addition of a recommended ionic strength and pH buffer to the distillate allows a direct determination of the fluoride concentration. The overall error was less than 0.5% and the method is sensitive to the 2-ppm level in biological samples. The method is simpler than the longer and more difficult AOAC method, but gives results which compare in precision. FUSION AND COLORIMETRICmethods for the determination of fluoride are either complicated or nonspecific. In applying these methods to a variety of biological samples ( I , 2), various difficulties have been experienced. The separation of fluoride from interfering substances, based on fusion decomposition followed by either distillation (3), or diffusion (4) is timeconsuming and requires very precise control to obtain reliable results. Hall (1) has recently found that when the temperature rises above 400 "C, some muffle furnaces cause serious and variable contamination of ashed specimens such as noted in previous studies (5). It is generally considered necessary to ash most materials at a temperature of about 600 "C. Most colorimetric methods proposed for the final determination of fluoride depend on the bleaching action on a particular organometallic dye complex (6). The greatest difficulty is the interference by foreign ions (7) which can also form stable complexes with either the metal or the fluoride ions present. When these methods are used for biological materials, fluoride must be separated from other elements to avoid these interactions. Thus the accuracy of such methods can be severely hindered by incomplete separations and there are significant reagent and apparatus blanks. A solid-state fluoride ion-selective electrode which responds to fluoride ion activity was recently developed by Frant and Ross (69, and has successfully been employed for a number of fluoride analyses in different samples (9-11). In the present study, the ion-selective electrode has been used to measure the fluoride concentration by an improved nonfusion p F method. This method has been applied to fluoride analyses of fish protein concentrate (FPC) and also to the raw material for this product.
(1) R. J. Hall, Analyst, 93, 461 (1968). (2) G. D. Ritchie, J. Assoc. OfJic. Anal. Chem., 51, 773 (1968). (3) M. A. Wade and S. S, Yamamura, ANAL.CHEM., 37, 1276 (1965). (4) L. Singer and W. D. Armstrong, Anal. Biochem., 10,495 (1965). ( 5 ) R. J. Hall, Proc. SOC.Anal. Chem., 3, 162 (1966). 22, 1190 (1950). (6) H. H. Willard and C. A. Horton, ANAL.CHEM., (7) N. T. Crosby, A. L. Dennis, and J. G . Stevens, Analyst, 93, 643 (1968). (8) M. S. Frant and J. W. Ross, Jr., Science, 154, 1553 (1966). (9) B. A. Raby and W. E. Sunderland, ANAL.CHEM., 39, 1304 (1967). (10) L. Singer and W. D. Armstrong, ibid., 40, 613 (1968). (11) L. A. Elfers and C. E. Decker, ibid., p 1658.
EXPERIMENTAL
Apparatus. An all-glass distillation apparatus with Claisen top fitted to a 3-neck 150-ml flask, was employed in preliminary distillations to separate fluoride ion from other compounds. A 150-ml separating funnel with a 24/40 joint and a 20 cm long dropping tube extending into the flask was set on the distillation head, Through the side neck of the still, the bulb of a 0-200 "C thermometer was immersed in the acid-sample mixture to within 5 mm of the bottom of the flask. An adapter tube was connected to the condenser. The tip of this tube was covered by 0.1N NaOH in the 250-ml polyethylene flask used to collect the distillate. The apparatus must be routinely washed with an acidic cleaning solution and hot 20% NaOH to prevent cross contamination. An Orion Model 94-09 solid-state fluoride selective electrode was used for this study. It consists of a single-crystal membrane of lanthanum fluoride doped with europium and cemented in an all-epoxy body containing an internal solution of 0.1N NaCl and 0.1N NaF. An Orion Model 401 Specific Ion Meter equipped with expanded MV scale and temperature and slope compensators was used to determine the fluoride concentration. Reagents. Water distilled twice in all-glass apparatus was used for this study. Merck reagent-quality sodium fluoride was further purified (12). A standard fluoride stock solution was prepared from a weighed amount in distilled water and stored in a polyethylene bottle. All other reagents were prepared from stock ACS-grade chemicals without further treatment, but each material was tested for fluoride content by adding sufficient known fluoride in each case to bring the level into the working range of the method. TISAB-1 (Total Ionic Strength Adjusted Buffer) was prepared by dissolving 340 g NaN03, 1 g sodium citrate, and 120 ml glacial acetic acid in 500 ml distilled water, and diluting it to 1 1. after adjusting the pH to 5.5 with 12N NaOH. TISAB-2 was made up similarly but without the sodium citrate. Samples. Samples A and B were FPC (fish protein concentrate) made on a pilot-plant scale from whole cod and cod fillets, respectively, by the Halifax isopropyl alcohol process in the Halifax Laboratory of the Fisheries Research Board of Canada (13). These finely powdered materials were kept in polyethylene bottles after being air dried in an oven at 40 "C for 24 hours. Sample C was a homogenate of whole herring made from four freshly killed fish. Sample D was prepared by homogenization of cod fillets. Both samples C and D were analyzed on a wet basis (ca. 80% water) and were stored in a freezer in closed containers until needed. Procedure. The sample, either 2-5 grams of FPC or 6-10 grams of fish homogenate, was placed in the still with a few acid- and alkali-washed glass beads. The digestion was carried out with 60 ml of 65% HClO, and 5 ml of 50% AgC10,. The still was heated by a Wood's metal bath on an electrical hot plate. Fluoride was separated by distillation while the temperature of the mixture was controlled between 140 to 150 'C by addition of distilled water from the dropping funnel through a fine adjustment glass stopcock. From 150 (12) J. J. Lingane, ANAL.CHEM.,39, 881 (1967). (13) H. E. Power, J. Fisheries Res. Board Can., 19, 1039 (1962).
VOL. 41, NO. 8, JULY 1969
1081
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to 200 ml of distillate was collected in 20 ml of 0.1N NaOH containing a few drops of phenolphthalein to indicate the basic condition of the distillate. The distillate was then diluted with distilled water to 250 ml in a volumetric flask as an unknown stock solution. Plastic-ware (polyethylene) was used for the following operations. Aliquots of 10, 20, and 40 ml of unknown stock solution were transferred to 50-ml volumetric flasks, 10 ml of TISAB-1 was added, and in the first two instances diluted to volume with distilled water. Four standards, in concentration of 20.0, 2.00, 0.20, and 0.020 pg/ml, were prepared by the same procedure from the standard NaF solution. Based on an estimate of the fluoride content of the unknowns, two standards with a concentration ratio of ten, were selected to calibrate the specific-ion meter. Fluoride concentration of unknown solutions was determined at pH 5.5 to 6.0 on the direct-reading scale for monovalent anions of the Orion meter. The rate of response of the meter depended upon the fluoride content and all readings were taken after stirring for three minutes. By using dilution factors depending upon the size of aliquots, the fluoride content of a sample, in ppm, was calculated from the average. Fluoride contents were also determined by a modified AOAC method (14). In this procedure 20 ml of 6N NaOH was used as the fusion reagent in a nickel crucible. The final fluoride determination was by a thorium-alizarin titration. RESULTS AND DISCUSSION
The low solubility product of crystalline LaF3 permits the use of this electrode over a wide pH range (15) in presence of many ligands that may form stable complexes with lanthanum ion, The Nernstian behavior of fluoride activity is good to 10-jM (0.19 ppm), while the electrode has usable response at least to 1 0 P M (0.02 ppm). However, the fluoride electrode response is also affected significantly by moderate con(14) "Official Methods of Analysis of Association of Official Agricultural Chemists," 10th ed., Washington, D. C., 1965, p 360. (15) K. Srinivasan and G. A. Rechnitz, ANAL.CHEM.,40, 509 (1968). 1082
ANALYTICAL CHEMISTRY
centrations of certain other ions. Other than a few qualitative investigations (7, 8, 16, 171, no detailed studies have dealt with the complicated effects of these interfering ions on the determination of fluoride activity. The effects of varying concentrations of the major ions in biological materials and of nickel and barium ions OR the response of the fluoride electrode are shown in Figure 1. All the data in this figure have been corrected for variable ionic strength (18). Obviously chloride ion can cause interference, but only when its concentration exceeds a certain limit. Hydroxide ion has been reported by Frant and Ross (8) as the only monovalent anion which will interfere. Nitrate and phosphate ions, even at a quite high concentration, are without effect on the fluoride determinations in pure systems. Ferric(III), and ferrous(I1) ions have important influences on the proposed analytical method, mainly because of complex ion formation (19). Fortunately, these difficulties can be avoided by eliminating most of these ions by distillation and complexing any which might have been entrained with citrate contained in the TISAB-I used for p F concentration measurements. With careful distillation, the citrate may not be necessary. Recently it has been reported that chemical interactions of calcium and magnesium with fluoride are especially important in fluoride determination of biological samples which usually contain relatively large amounts of Ca and Mg in comparison to fluoride (10, 16). Accordingly, it becomes obvious why a simple and direct procedure (10) for fluoride determination with an ion-selective electrode could not be applied to all biological specimens without provision for a preliminary separation of these ions. Since the fluoride electrode is quite sensitive to the activity of fluoride provided the interfering ions are below the critical concentrations shown in Figure 1, one nonfusion distillation is satisfactory to meet the requirements of the present analyses. Considerable amounts of phosphate distilled over *ith the fluoride but the amount from the samples examined in this (16) J. D. Johnson and E. D. Daugherty, Department of Environmental Science and Engineering, University of North Carolina (Chapel Hill), U. S. A., Publication No. 177 (1968). (17) L. H. Andersson and B. Gelin, FOA (Foersvarets Forskningsanst) Rep., 1, 5 (1967). (18) P. Debye and E. Hiickel, Phys. J., 24, 185 (1923). (19) K. Srinivasan and G. A. Rechnitz, ANAL.CHEM.,40, 1818 (1968).
Table I. Reproducibility of Nonfusion Distillation pF Method for Fluoride Determination with Internal Standards Added fluoride recovered Number of f S.E. at 150 =k 3 "C. Fluoride added, pg aliquots analyzed Sample size, g 100.05 i 0.06 None 1500 6 Sample A (239.9 ppm F-) 100.10 1. 0.10 2.023 500 99.95 f 0.15 2.046 750 99.68 f 0.12 2.948 250 1.279 99.77 f 0.08 lo00 99.88 i 0.12 5.160 500 Av 99.91 i 0.16 Sample C (28.4 ppm F-) 3 99.72 f 0.06 6.459 500 100.18 i 0.08 3 9.800 250 3 99.87 i 0.12 10.166 100 Av 99.92 f 0.26 Table 11. Percentage of Total Fluoride Recovered by Nonfusion Distillation at Several Temperatures Recovery of loo0 pg F-, Volume of distillate, ml 20 40 60
100 150 200
From Sample A (4.1667 g) 130 f 3 "C 24.0 44.4 56.8 74.0 83.6 90.1
140 f 3 "C 30.4 57.3 70.2 86.6 94.8 99.9
160 i 3 "C 48.3 84.8 98.5 99.9 100.0 100.1
140-50 "C 33.6 62.1 78.1 95.1 100.0 100.0
From F-standard 140-50 "C 45.0 81.7 93.5 99.3 100.0
Table 111. Determination of Fluoride in Various Samples by Nonfusion pF Method F-content (ppm) calculated from Sample size, g Sample A 1.954 3.014 4.890 2.023 Sample B 2.058 4.101 5.062 4.109 Sample C 6.540 8.009 10.271 10.066 Sample D 7.011 9.113 10.370 10.109
F-Standard, pg
500
100
100
40-ml alq
Av
240.1 240.2 239.7 239.5 239.4 239.1 240.9 240.7 Over-all av 239.9 =t0.58 (std dev)
240.0 239.8 239.2 240.6
240.1 239,6 239.2 240.7
21.3 21.4 21.2 21.3 21.3 21.3 21.4 21.5 Over-all av 21.3 i 0.11 (std dev)
21.3 21.1 21.2 21.4
21.3 21.2 21.3 21.4
28.4 28.6 28.6 28.6 28.3 28.2 28.4 28.4 Over-all av 28.4 1. 0.14 (std dev)
28.5 28.5 28.3 28.5
28.5 28.6 28.3 28.4
10-ml alq
... ...
50
20-ml alq
2.40 2.30 ... 2.35 2.34 2.37 Over-all av 2.36 i.0.03 (std dev)
study were still far below the limit which would affect the determination. The chloride can be effectively retained in the still by adding sufficient AgC104. Sodium fluorosilicate has been suggested for use as a standard for fluoride analyses (14). It has also been postulated that some fluorosilicates may be formed as the result
2.35 2.31 2.40 2.36
2.36 2.32 2.38 2.36
of reaction with the glass in the distillation equipment ( 2 0 ) . The response of the ion-specific electrode to sodium fluorosilicate was compared with that of sodium fluoride with the results shown in Figure 2. Only a t very low concentrations, (20) M. S . Frant, Plating, SO, June (1967). VOL. 41, NO. 8, JULY 1969
1083
Table IV. Comparison of Results of Nonfusion pF Method and Modified AOAC Method F-content (ppm f std dev) by pF method by AOAC method pF/AOAC 239.9 f 0.58 239.5 f 1.40 1.0017 21.3 f 0.11 21.1 + 0.38 1.0095 28.4 f 0.14 28.2 f 0.45 1.0071 2.36 f 0.03 2.32 =t0.29 1.0172 below lO+M, is there any deviation in the electrode response. The slope of the line is 72 mV per pF unit at 21 OC. These results show that there is no significant bias inherent in the distillation-electrode procedure such as had been encountered in a previous study (3). The accuracy and reproducibility of the revised method have been studied and the results are listed in Tables I and 11. Errors less than OSy0 can be expected when the distillate volume is between 150 and 200 ml and the still temperature is kept within the 140-150 OC range. The percentages of total fluoride recovery in the distillate at different tempeiatures are compared in Table 11. There was a slower rate of distillation of the fluoride from the biological sample as compared to the NdF standard. This slower rate is probably due to the more complex system present in the biological sample distillation (21). (21) R. Valach, Talunru, 8, 629 (1961).
Since the total ionic strength has been established at an essentially stable level in this method by use of a buffer, the activity coefficient of the fluoride can also be considered constant and the electrode responses can be translated directly into concentrations. The results in Table 111 show the high degree of reproducibility of the determinations as well as determinations on separate samples prepared individually. Comparison of analyses between the nonfusion distillation p F method and the modified official AOAC procedure are given in Table IV. Generally, the results by the method described here are more precise and are slightly higher than those obtained with the official method. This procedure using a solid fluoride membrane electrode is simpler and faster than the present official method. Also, it should be applicable to the determination of fluoride in most biological materials even though they contain a wide range of potentially interfering ions. ACKNOWLEDGMENTS
The constant advice and encouragement of Dr. R. G . Ackman is gratefully acknowledged. Mr. C. A. Campbell prepared the samples of FPC. RECEIVED for review March 10, 1969. Accepted April 17, 1969. Project supported in part by the Industrial Development Service of the Department of Fisheries of Canada. ~~
~
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Nitrogen and Oxygen Compound Types in Petroleum Total Analysis of an 850-1000
O F
Distillate from a California Crude Oil
L. R. Snyder Union Oil C o . of California, Union Research Center, Brea, C a l g . The oxygen and nitrogen compound types (heterocompounds) present in the 850-1000 O F fraction of a California crude oil were analyzed by means of a standard separation scheme, high resolution mass spectrometry, and other techniques. All compound types present in other than trace amounts were accounted for: their molecular structures were determined and their concentrations were measured. The major heterocompound types present in this sample (> 0.5% wt) are similar to those reported previously in lower boiling distillates from the same crude oil: indoles, carbazoles, benzcarbazoles, pyridines, quinolines and higher benzologs, pyridones and quinolones, dibenzofuranes, naphthobenzofuranes, phenols, carboxylic acids, and aliphatic ketones and esters. In addition, several minor heterocompound types were encountered for the first time: dinaphthofuranes, aromatic compounds containing both nitrogen and sulfur, oxygen and sulfur, or two oxygen atoms per molecule, as well as certain new aliphatic heterocompound types. Provisional structures have been assigned to several of these new compound types. Total heterocompounds in the various distillates from the present crude oil comprise 3% wt of the 400-700 O F distillate, 13% w t of the 700-850 O F distillate, and 21% wt of the 850-1000 O F distillate.
PRECEDING PAPERS ( I , 2) have described the complete, detailed analysis of the nitrogen and oxygen compounds (heterocompounds) in the 400-700 and 700-850 O F distillates from a California crude oil. A standard separation scheme was 1084
ANALYTICAL CHEMISTRY
used in conjunction with spectral and chemical analyses of the resulting fractions. These studies have now been extended to the next higher boiling fraction from the same crude oil: the 850-1000 O F distillate. This fraction shows an expected increase in complexity; several new heterocompound types were observed in addition to the same compound types found in lower boiling fractions. The analysis of the 850-1000 O F fraction proved more difficult than in the case of lower boiling fractions because of its greater complexity and higher molecular weight. This required minor modifications in the procedure used previously to analyze the lower boiling distillates. In addition, our experience with the lower boiling fractionsand analytical data from these fractions-has served to compensate for some of the shortcomings of the present separation and analysis procedures. The present study adds to our collection of spectral data for the heterocompound types in these various distillates, and it provides new information on how these heterocompound types can be separated from each other and from other sample components. Hopefully these data can serve as the basis for routine procedures for the rapid analysis of any petroleum fraction in terms of heterocompound type. (1) L. R. Snyder, B. E. Buell, and H. E. Howard, ANALCHEM.,40,
1303(1968). (2) L. R. Snyder, ibid., 41, 314 (1969).
