Reverse Extraction Technique for the Determination of Fluoride in Biological Materials P. Venkateswarlu Department of Biochemistry, The Medical School, University of Minnesota, Minneapolis, Minn. 55455
A new approach has been developed for the isolation and concentration of fluoride from a variety of materials. It involves extraction of fluoride, from an acidified sample, as fluorosilane into an immiscible organic solvent, followed by reverse extraction as fluoride ion into an alkaline solution. Starting with a 2-ml sample, it is possible to concentrate the fluoride into 30-40 pl of the back-extract. To take advantage of the resulting 30- to 50-fold gain in fluoride concentration and to determine fluoride in a few microliters of the back-extract, a simple hanging drop fluoride electrode assembly, applicable to 2.5 picomoles of fluoride in 5-pl samples has also been devised.
Bock and Semmler ( 1 ) proposed g a s c h r o m a t o g r a p h y and mass s p e c t r o m e t r y for the determination of fluoride, following its e x t r a c t i o n as a fluorosilane, into an organic phase. Based on this suggestion, Fresen, Cox, and Witter ( 2 ) developed a g a s c h r o m a t o g r a p h i c procedure for the determination of fluoride i n biological materials. The present s t u d y w a s undertaken to render the extraction technique amenable to the determination of fluoride with the fluoride specific ion electrode, which is s i m p l e r and less expensive than g a s c h r o m a t o g r a p h y or mass spect r o m e t r y . The use of the electrode is feasible only if fluoride, a f t e r e x t r a c t i o n as fluorosilane f r o m the biological material i n t o a w a t e r immiscible organic phase and thus virtually freed f r o m the bulk of interfering substances in the s a m p l e , could be q u a n t i t a t i v e l y reconverted t o fluoride ion in an aqueous phase. This has b e e n accomplished b y back-extracting the fluorosilane w i t h an alkaline aqueous solution, w h e r e b y the fluorosilane is decomposed and the fluoride ions are released into the aqueous p h a s e , as shown in Figure 1. Fluoride is then d e t e r m i n e d w i t h the fluoride electrode. T w o procedures, involving the use of the fluoride electrode, for the d e t e r m i n a t i o n of fluoride, following its isolat i o n b y t h e reverse e x t r a c t i o n technique, are described in this paper.
EXPERIMENTAL Reagents, Extractant: 2.5mM diphenylsilanediol (DPSD) in toluene is prepared by dissolving 540 mg DPSD in 1000 ml toluene. Perchloric acid 70% (G. Frederick Smith Chemical Company) was used. Histidine buffer, pH 6.1: 7.75 grams L-(+)-histidine are suspended in water and 1N hydrochloric acid is added dropwise with continuous stirring until pH 6.1 is attained. The volume is made to 50 ml with water to obtain 0.5M histidine buffer, pH 6.1, which is appropriately diluted for preparing 0.25M, O.lM, 0.0625M, and 0.05M histidine buffer solutions. Sodium fluoride stock solutions: 0.1M fluoride solution is prepared by dissolving 4.2 grams sodium fluoride (analytical reagent
(1) R. Bock and H. J . Semmler, Fresenius' Z. Anai. Chem., 230, 161 (1967). (2) J. A . Fresen. F . H . Cox, and M . J . Witter, Pharm. Weekbiad.. 103, 909 (1968).
878
A N A L Y T I C A L C H E M I S T R Y , V O L . 46, N O . 7 , J U N E 1974
grade) in 1 liter double distilled water. This solution is appropriately diluted to obtain fluoride stock solutions 4 to 40pM. Apparatus. The equipment used included 15-ml polypropylene stoppered centrifuge tubes (Nalgene 17C); 4-ml polypropylene disposable tubes with caps (Falcon tube, 2063); 10-ml polystyrene disposable tubes (Falcon tube, 2001); Corning research p H meter, model 12; and an Orion fluoride ion electrode, model 94-09A. Cleaning Methods. Re-used 15-ml polypropylene centrifuge tubes are cleaned successively with Sparkleen (Fisher), Chems o h (Mallinckrodt) and 30% nitric acid, with several rinsings inbetween with de-ionized water and finally with double distilled water. A more rigorous alternate procedure employed when the samples are expected to have very low fluoride contents, involves washing the tubes with 20% perchloric acid and 50 mg % DPSD in benzene or toluene, followed by rinsing with acetone. Four-ml polypropylene tubes (Falcon tube, No. 2063) are used fresh from the packing case without additional cleaning and are discarded after use. Fluoride blank determinations are carried out in each set of analyses as a check on fluoride contamination of the tubes. Procedures. A . Reverse Extraction, Fluoride Electrode Procedure (Macro Method). Four ml of extractant is layered on the top of 2 ml of the sample (serum) in a 15-ml polypropylene centrifuge tube. One ml ice-cold concentrated perchloric acid is added and the tube stoppered promptly. The contents are vigorously shaken for 1 hour on a laboratory shaker, whereby the fluoride in the aqueous phase is transferred as a fluorosilane into the organic phase (first extraction, Figure 1). The tubes are centrifuged for 10 minutes at 3000 rpm. Protein, if present in the sample, is partly precipitated at the bottom of the tube and partly at the interphase of the aqueous and nonaqueous layers. Three ml of the extract (top layer) containing fluorosilane, are carefully withdrawn and transferred to another 15-ml polypropylene tube. Fifty p1 2.5N NaOH are added. The tube is stoppered and shaken for 1 hour, whereby the fluoride is back-extracted from the organic phase into the 50-pl NaOH solution (reverse extraction, Figure 1). The tube is centrifuged for 5 minutes at 2000 rpm. The organic layer is removed by suction and the sodium hydroxide solution is washed twice with 2 ml of benzene which is also removed by suction. The residual benzene is removed by placing the tubes in an evacuated desiccator for 5 minutes. The air admitted into the desiccator for breaking the vacuum is drawn through scrubbers containing 5 N thorium nitrate solution and 5 N sodium hydroxide solution. The back-extract is neutralized with 50 p1 2.5N hydrochloric acid and buffered with 500 p1 0.05M histidine buffer (pH 6.1). The contents are transferred to a 10-ml polystyrene tube, a small magnetic stirring bar is dropped in, and the fluoride electrode introduced. Contact with the reference electrode, immersed in a few milliliters of saturated potassium chloride solution, is made through an agar bridge (4% agar in 0.M sodium chloride and 0.05M histidine buffer, p H 6.1). The fluoride standards (0.5. 1.0, 1.5, 2.0, and 2.5 mmoles F/600 pl) are prepared by adding 100 p l each of the sodium fluoride stock solutions (5, 10, 15, 20, and 25 pM F ) to a mixture of 50 p l of 2.5N sodium hydroxide, 50 PI 2.5N hydrochloric acid and 400 PI 0.0625M histidine buffer. For recording the potentials of the electrode with the standards and samples, a response time of 30 minutes is followed. The solutions exposed to the fluoride electrode are continuously stirred. B Reverse Extraction, Hangmg Drop Electrode Procedure (Micro Method). The procedure is essentially the same as in procedure A. Both the first extraction and the reverse extraction are carried out in 4-ml stoppered polypropylene tubes. The first extraction is carried out employing 1 ml sample, 1 ml extractant, and 0.5 ml concentrated perchloric acid. The reverse extraction is carried out by shaking 800 pl of the toluene extract with 100 concentrated ammonium hydroxide. Neutralization of the backextract with HCI as in procedure A and consequent formation of salt are avoided by removing the excess ammonia by placing
Immiscible
R,SiOH % R
Organic Phase
3
S
i
F
_________
~
,
g
____ ._ _ _ ._ ~. ____
IO-~M
*-
Firs: Ext roction
=
-
0l9Oppm Reverse Extraciion
c
*
Figure 1. Schematic representation of the principles of reverse extraction technique for isolation of fluoride. R stands for acylor aryl- groups
0 .c
e
l
"pF" Readings 4i4
4 ;
4,8
5;
5,2
5,4
Macro Method
Macro Mefhod
c
c
Q)
10-6M O.O/9ppm
c t
J \Micro Method pF Reodngs
4I0
60 I
80 I
lo0 i
120 I
140 I
Milli Volts Figure 3. Calibration curves, millivolt, and "pF" readings YS. concentration, for macro and micro methods for determination of fluoride with specific ion electrode. The "pF" readings are those taken from the expanded pH scale of the instrument
v
Figure 2. Hanging drop fluoride electrode assembly
the tubes containing the back-extract in a continuously evacuated desiccator for 60 minutes. As a consequence, the volume of the back extract is reduced to 30-40 pl. Five pl 0.25M histidine buffer, pH 6.1, are added to a 20-p1 concentrated back-extract before measurement with the hanging drop fluoride electrode. The volume of the back-extract is determined by the difference in weight of the tube with and without the back-extract. The latter is determined after wiping out the excess back-extract with a cotton swab, soon after the 20-pl aliquot has been removed. I t is unsafe to depend on the weight of the empty tubes prior to back-extraction; these tubes tend to gain in weight during the course of the experiment depending on humidity and possibly other factors. The fluoride standards, (2 to 20 pM F), are made by adding 100 p1 of each of the sodium fluoride stock solutions (4to 4 0 p M F) to 100 p1 0.1 M histidine buffer, pH 6.1, and unlike the standards in procedure A, do not contain sodium chloride. The hanging drop fluoride electrode assembly (Figure 2) is selfexplanatory except for the following points. The agar bridge contains 0.4% agar in 0.05M histidine buffer, pH 6.1. The 5 - p l sample applied to the crystal of the electrode occasionally tends to drift with the result that contact with the agar bridge is broken. T o overcome this problem, the following improvisation is made. A small square of Parafilm is stretched taut over the crystal of the fluoride electrode and tightly secured by adhesion. A small hole is made in the Parafilm with a sharp plastic probe exposing a small surface of the lanthanum fluoride crystal. The end of the electrode is then pressed evenly against a pad of filter papers covered with a strip of glazed weighing paper to eliminate all crevices between the crystal and the Parafilm into which the sample may otherwise be drawn by capillary action. After assembling the hanging drop fluoride electrode, the agar bridge is secured in position with a strip of Parafilm wrapped around the nozzle, the sleeve, and the bridge. A completely closed system, obviating the evaporation of the 5-p1 sample is thus achieved. Between reading
samples, the electrode is wiped gently with a cotton sponge, without disturbing the Parafilm cover and rinsed with 5 pl of the "blank" solution (0.05M histidine buffer, pH 6.1) and with a 5-pl aliquot of the following sample. Also, the droplet of the previous sample adhering to the tip of the agar bridge is removed by wiping with a cotton swab. Reading times with the electrode are kept the same for all the standards and the unknowns. For weak fluoride solutions (IpM), a maximum of half an hour has been employed. The calibration curves, millivolt and "pF" readings us. fluoride concentrations, for both the micro and macro procedures, are shown in Figure 3. The "pF" readings are those from the expanded pH scale instead of the millivoltage scale of the Corning research pH meter, model 12. The sensitivity of the readings from both scales is greater with the microprocedure than with the macroprocedure. This is due to the lower ionic strength in the final buffered back-extract obtained in the microprocedure, in which the ammonia from ammonium hydroxide used for reverse extraction, is removed by evacuation. In the macroprocedure, however, the sodium hydroxide employed for reverse extraction is neutralized with acid and an equivalent amount of salt is formed, increasing the ionic strength of the back extract. If ammonium hydroxide were used for reverse extraction in the macroprocedure, better sensitivity could be expected. The response of the electrode diminishes markedly below 10-6M fluoride concentration. The reverse extraction, coupled with the hanging drop electrode method provides a means for isolation and concentration of fluoride from samples so as to attain fluoride concentrations greater than 10-6M, thus ensuring reliable measurements with the electrode. All fluoride values reported in this paper are based on electrode measurements at fluoride concentrations greater than 10-6M, and "pF" readings from the pH scale. which gives a larger scale deflection for a given change in fluoride concentration than the millivoltage scale, are routinely used for constructing the calibration curves.
RESULTS AND DISCUSSION Fresen, Cox, and Witter ( 2 ) extracted fluoride as trimethylfluorosilane (bp 16.4 "C) with benzene (bp 80 "C) for gas chromatographic determination of fluoride. However, in studies involving ISF, it was found that trimethylfluorosilane partly escaped from the organic phase on standing. We, however, obtained good recoveries, 99.8 f 0.59 (SE) %, N =15, employing trimethylchlorosilane in ANALYTICAL CHEMISTRY, VOL. 46, NO. 7, JUNE 1974
879
Table I. Comparison of Results of Fluoride Analyses by a Diffusion Technique and the Reverse Extraction Procedure % F - in bone ash High fluoride intake rat
Low fluoride intake rat
Reverse extraction, mean i S.E. ( N j a
0.4010 f 0.0032 (6)
0.400p =!= 0.0057 (6) TMCSb (toluene) 0.3950 =t 0.0157 (5) TMCS (benzene) 0.0032 f 0.0001 (6) TMCS (toluene)
0.0030 0.0031
pM F- in human urine Sample 1 Sample 2 a Cerium-alizarincomplexan
Diffusion, mean i S.E. (N)‘
44.2 i 0 . 8 6 (6) DPSDc (toluene) 57.8 i 0 . 9 7 (6) DPSD (toluene)
46.3 i 0 . 8 4 (4) 50.0 =t 0.16 (6)
method ( 3 ) used in t h e final step in both of the procedures. TMCS: Trimethylchlorosilane.
Table 11. Analyses of Animal and Human Blood Sera by the Reverse Extraction, Fluoride Electrode Technique (Procedure A)
DPSD: Diphenylsilanediol.
Table 111. Recovery of Radioactive Fluoride (’SF) Added to Biological Materials by the Reverse Extraction Technique.
$3 Recovery of added F, Sample
r M F-, mean i S.E.
(N)
Bovine serum 1 0 . 5 3 f 0.016 Bovine serum 2 0 . 5 8 =t 0.026 Rabbit serum 1 . 7 9 f 0.021 Human serum 1.32 f 0.126
(7) (8) (7) (3)
0.53 p M , mean i S.E.
99.3 100.7 99.2 98.7
(N)
f 1 . 3 7 (8) f 1 . 7 1 (4) f 3 . 5 2 (7) i 3 . 9 5 (6)
benzene for extracting 50 nmoles of fluoride and by promptly carrying out the reverse extraction. To avoid loss of fluoride or excessive reduction in volume of the organic phase through solvent evaporation, particularly while handling a large number of samples, it was found best to extract fluoride as diphenyldifluorosilane (bp 157 “C) with toluene (bp 110 “C). The recovery of 10 nmoles fluoride from aqueous standard solutions was 99.8 f 1.39 (SE) %, N = 17. Fluoride in samples of bone ash and urine was determined spectrophotometrically following isolation of fluoride by the reverse extraction technique and also by the Baumler-Glinz diffusion technique ( 3 ) which is a modification of the original diffusion technique developed by Singer and Armstrong ( 4 ) . The agreement between the results of fluoride analysis obtained by both methods (Table I) indicates the reliability of the reverse extraction technique. Although fluoride in bone ash and urine samples can be determined by reverse extraction and spectrophotometric procedure, fluoride in blood serum so extracted is not amenable to determination by spectrophotometric procedures, because of the low levels of fluoride and also because of the carry-over, from the serum to the back-extract, of traces of substances which interfere with the spectrophotometric procedures. Instead, the fluoride electrode was employed advantageously in this situation, with a two- to threefold gain in the concentration of fluoride compared with that in the original sample. Recoveries of 0.53 nmole of fluoride added per milliliter to bovine, rabbit, and human sera are satisfactory, ranging from 99.2 to 100.7% (Table 11). Results shown in Table I11 indicate that the recoveries of ISF incorporated in vivo into rat body fluids and tissues (plasma, kidney, muscle, liver) and of 18F added to samples of serum, urine, extracts of pea plant, and to washings of products of combustion in an oxygen bomb (Parr, Model 1108) of rat liver and muscle are all satisfactory with the exception of liver tissue. The studies with 18F were carried out to obtain a check of fluoride recoveries (3) J Baumler and E Glinz, M i f f Geb Lebensmittelunters Hyg 250 (1964) (4) L Singer and W D Armstrong. Anal Chem 26, 904 (1954)
880
L ; Recovery, mean i S.E. (N)
Biological material
55,
A N A L Y T I C A L C H E M I S T R Y , V O L . 46, NO. 7, JUNE 1974
Bovine serum Bovine serum (plus 0 . 5 3 pM F-) Human urine Extract of pea plant R a t plasmab R a t kidneyb R a t muscleh R a t liverb R a t liver (Oxygen-bomb washings) R a t muscle (Oxygen-bomb washings)
97.8 f 0.55 97.1 f 0.47 9 9 . 7 f 0.91 100.1 f 0 . 8 1 100.5 i 0 . 0 5 95.5 0.96 98.5 f 0.96 8 8 . 2 i. 0 . 4 8 96.3; 97.2 98.5; 9 6 . 4
(4) (4) (4) (7) (4) (4) (4) (4)
Procedure A, see text for details. A 300-g rat was sacrificed 1 hr after intraperitoneal injection of radioactive fluoride ( 1 . 5 ml containing 1 . 6 mg F -). In all other cases, radioactive fluoride was directly added to t h e samples.
Table IV. Comparison of Results of Fluoride Analyses of Serum by Reverse Extraction and Other Methods
Sample
Calcium phosphate adsorption ( 5 ) # M F - = S.E. (N)
Bovine 0 . 7 f 0.037 (7) serum Human 1 . 3 & 0.063 (4) serum
Reverse extraction (Procedure A) pM FS.E. (N)
+
Ultrafiltration, F electrode (6)
pM F -
0 . 6 f 0 . 0 1 1 (4)
0.6
1 . 3 f 0.013 (3)
1.5; 1 . 5
independent of errors of analysis by colorimetric and electrode techniques. Serum samples were analyzed for fluoride by (a) calcium phosphate adsorption technique ( 5 ) , (b) fluoride electrode measurement of the ultrafiltrate ( 6 ) , and (c) the reverse extraction technique (Procedure A). Results obtained by all three procedures are similar suggesting that the silanol extractable fluoride occurs as ionic (plus ionizable) fluoride in normal sera (Table IV). However, it should be pointed out that the extraction is carried out a t a very low p H (about 20% HC104) and, should the sample being analyzed contain any acid labile organofluoro compounds, such as those shown by Taves et al. (7) to be present in plasma of subjects receiving methoxyflurane anesthesia, the fluoride measured by the extraction technique would be expected to measure the total of ionic fluoride and the “acid labile fluorine” in the sample. That this indeed is the case is shown by the results in Table V. (5) P. Venkateswarlu, L. Singer, and W . D. Armstrong. A n a / . Biochem.. 42, 350 (1971) (6) L. Singer and W. D.Armstrong, Biochem. Med.. 8, 45 (1973). (7) D. R. Taves, 6 . W . Fry, R . B. Freeman, and A. J. Giles, J . Amer. Med. A s s . . 214,91 (1970)
Table V. Analyses of Ionic and Nonionic Fractions of Fluoride in Plasma of Rats That Had Methoxyflurane Anesthesia by Intraperitoneal Injection a Week Earlier Ionic fluoride plus F - from acid labile F-metabolites p M F - i. S.E. ( N )
Procedure"
a. Diffusion with HCIOd a t 60 "C, 22 hr, Ce-alizarincomplexan method (3)
b. Oxygen bomb, F-electrode c. Silanol extraction, F-electrode (Procedure A)
Table VIII. Determination of Fluoride in Rat Tail Tendon. by Reverse Extraction, Hanging-Drop Electrode Technique (Procedure B)
Group
F content drinking of water, ppm
A
0
B
10
C
50
121 =k 1.26 (3) 113 & 3 . 0 5 (4) 118 (1)
" Calcium phosphate adsorption technique applied t o t h e same plasma showed 1 4 . 7 fiM fluoride which is evidently more nearly t h e true ionic fluoride content.
F - per
g
Rat No.
Sample size, mg, dry w t
F - found, nmoles
d r y wt, nmoles
1 2 3 4 5 6 7 8
55.8 52.2 47.9 56.7 50 . O 55.3 50.5 55.4
0.13 0 .os 0.16 0.33 0.26 0.45 0.73 1.oo
2.33 1.53 3.34 5.82 5.20 8.14 14.46 18.05
a Weanling rats raised on low-fluoride diet for two months and supplemented with fluoride in drinking water as indicated.
Table VI. Comparison of Sensitivity of Hanging-Drop Electrode Procedure with That of the Conventional Fluoride Electrode Procedure Volume of sample employed
Procedure
Conventional procedurecJ Reverse extraction, hanging-drop electrode 'I
F - concentration
-
Absolute amount of F - in the sample employed
_
_
_
~
2 ml
0,0095 ppm
0 . 5 pM
19 ng
1. O nmole
5 P1
0.0095 ppm
0.5
0.0475 ng
0.0025 nmole
p M
-
Both the reference and the fluoride electrodes are immersed in the test sample placed in a n appropriately small beaker.
Table VII. Determination of Fluoride i n Rat Epiphyseal Cartilage (Tibia) by Reverse Extraction, Hanging-Drop Electrode Technique (Procedure B) F - per
Group
Normal weanling rats (Fed normal diet) Hachitic rats (Fed synthetic diet)
Hat No.
Sample size, d r y weight, fig
Fluoride found, nmoles
1
329.3 Pa 195.5 H 301.3 P 225.0 H 125.9 H 335.8P 291.7 H 102.6 P 108.4H 144.6 P 163.7 H
0.63 0.68 1.13 0.77 0.36 0.13 0.07 0.07 0.15 0.06 0.03
2 3 4 5 6
mg d r y
1.91 3.48 3.75 3.42 2.86 0.39 0.24 0.68 1.38 0.41 0.18
wt, nmoles
' P: Proliferating cartilage. H: Hypertrophic cartilage.
It is to be pointed out that diffusion of sera samples, either overnight or for shorter time periods, for determination of "total" fluoride (ionic plus acid labile fluorine) can be replaced by the more rapid silanol extraction. Prior to extraction, the acidified sample (final concentration 20% HC104) in a 15-ml polypropylene centrifuge tube was placed in a water bath for 2 hours a t 70 "C for the release of the entire acid-labile fluorine. In experiments using the same conditions no loss of 18F was observed. Obviously, depending on the degree of acid lability of the fluorocompound, one needs to determine the duration and conditions of acid treatment of the sample preparatory to extraction. The hanging drop electrode assembly permits measure, an ment of fluoride in samples as small as 5 ~ 1containing absolute amount of fluoride no greater than 2.5 picomoles (0.0475 ng F). Samples smaller than 5 ~1 have also been
employed. A comparison of sensitivity of the hanging drop electrode procedure with that of direct application of the fluoride electrode to solutions is shown in Table VI. For measurement of fluoride in small sample volumes (5 to 20 ~ 1with ) the fluoride electrode, the hanging drop electrode technique, which utilizes the readily available fluoride electrode, is simpler than the other published procedures which require reconstruction of the fluoride electrode for use in an inverted position ( 8 ) , use of two fluoride electrodes for null point potentiometry ( 9 ) , or special fabrication of a fluoride microelectrode using a tiny crystal of lanthanum fluoride (IO). Errors due to progressive dilution of micro samples with saturated solution of potassium chloride leaking from the reference electrode or from the combination fluoride electrode (Orion Research Inc., model 96-09) are obviated with the use of the hanging drop fluoride electrode. The hanging drop electrode technique could be readily extended to the measurement of fluoride in solutions other than the back-extracts and, also possibly. to determination of other ions, employing specific ion electrodes with a construction similar to that of the fluoride ion electrode. The reverse extraction and hanging drop electrode techniques were employed to determine fluoride in pooled microtome sections of rat tibia epiphyseal plates (kindly made available by P. K. Dixit, Department of Anatomy). The results are shown in Table VII. The observation of lower fluoride levels in rachitic cartilage made in this study is not to be interpreted as resulting from the rachitic condition of the rats, because the fluoride content of the synthetic rachitogenic diet was found to be very low, 0.8 ppm F, compared to 50 to 80 ppm F generally found in most commercial rat food. Employing these techniques, in another study, the fluoride contents of tail tendon of rats raised on a low fluoride diet (0.5 pprn F) for two months ( 8 ) R A Durst and J K Taylor, Ana/ Chem 39, 1483 (1967) (9) R A Durst Ana/ Chem 40, 931 (1968) (10) R A Durst Anal Chem 41, 2089 (1969)
A N A L Y T I C A L C H E M I S T R Y , V O L . 46, NO. 7 , J U N E 1 9 7 4
881
following weanling and concomitantly receiving 0, 10, and 50 ppm F in the drinking water, were determined (Table VIII), The absolute amount of fluoride per sample involved in these studies (Tables VI1 and VIII) ranges from 0.03 to 1.13 nmoles. It would not be possible to determine fluoride in such small amounts by the standard diffusion technique (11) which has a blank of 0.00 to 0.08 Fg (i.e., 0.0 to 4.2 nmoles F) or by the modified rapid diffusion procedure (12) with a blank ranging from 0.00 to 0.52 nmoles F. The reverse extraction, hanging drop electrode technique has the advantage of having a low and less variable blank, 0.2 0.013 (SE) nmoles F, a feature which has made measurement of nmole quantities of fluoride possible. The chief feature of the reverse extraction technique is that it offers a new approach with potential advantages over the conventional distillation or diffusion procedures
*
(11) L. Singerand W. D. Armstrong, Anal. Biochem.. 10, 495 (1965) (12) D. R . Taves, Talanta, 15, 969 (1968).
for the isolation of fluoride from inorganic and organic materials. The advantages are the simplicity of the procedure, very low fluoride blank, and the very high degree of concentration of the isolated fluoride not hitherto possible to achieve. An additional application of the reverse extraction technique could be in obtaining highly concentrated preparations of radioactive fluoride free from other radioactive contaminants formed concomitantly in the course of production of the radioactive fluoride.
ACKNOWLEDGMENT I wish to express my sincere appreciation to Wallace D. Armstrong for drawing my attention for the first time to the extraction of fluoride as fluorosilanes, whereupon the reverse extraction technique described in this paper was conceived. I gratefully acknowledge his deep interest, and very helpful discussions throughout these investigations. Received for review November 6, 1973. Accepted February 6, 1974. These studies are supported by Grant DE-01850 from the National Institutes of Health, Bethesda, Md.
Analytical Method for the Determination of Less than Five PPM of Sulfur in Nickel Keith E. Burke The International Nickel Company, Inc., Paul D. Merica Research Laboratory, Sterling Forest, Suffern, N. Y. 10901
The high purity of commercial nickel has necessitated the development of an analytical technique for the separation and determination of less than five ppm of sulfur: Ten grams of nickel are dissolved at room temperature, in a solution 0.9M in both cupric potassium chloride and hydrochloric acid. The resulting residue contains all of the sulfur. The residue is separated on a sulfur-free filter, washed, dried, and burned in a stream of oxygen to convert all of the sulfur to sulfur dioxide. The resulting sulfur dioxide is titrated with a standard solution of potassium iodate. The proposed analytical technique gives accurate results for the determination of sulfur in the range from 0.2 to 20 ppm. The precision (a) is f0.7 ppm for the 2-ppm level. Some nickel has a sulfur content of less than 1 ppm. The method is also applicable to aluminum, iron, cobalt, and copper, as well as their alloys.
There are other applicable techniques, such as the evolution ( 3 ) or methylene blue ( 4 ) which have been discussed in detail (2, 5 ) . The Meineke Reagent (6) has been used for over a hundred years to determine macro amounts of sulfur. Now it has been applied, in this study, for the determination of trace 'amounts of sulfur. Once the sulfur has been concentrated it is determined by the combustion-titration method. The Meineke method uses a solution of the double salt, cupric potassium chloride CuCl2 .2KCle2H20, in dilute hydrochloric acid, to dissolve the sample and leaves a residue containing all of the sulfur, in part as copper sulfide. The reactions are as follows if it is assumed that all of the sulfur is present as nickel sulfide and the latter is slightly soluble in dilute acid.
+ CuCl, = NiCl, + Cuo + WCl = NiCl, + H,S H2S + CuC1, = CUS) + 2HC1 NiO
NiS
An analytical method is required for the determination of less than 5 ppm sulfur in nickel. The sulfur content of high purity nickel cannot be accurately determined by existing methods. Existing methods are capable of accurate determination down to the 10- or 20-ppm level by the rapid combustion-titration ( I ) procedure. The combustion-titration method has been used for the 3-ppm level, by using a 3-gram sample weight ( 2 ) . Larger sample weights are incompatible with the existing combustioninduction furnaces; therefore, accurate analyses at levels of less than 5 ppm require the preliminary separation of sulfur. (1) K.E. Burke, Anal. Chem., 39, 1727 (1967). (2) C. L. Lewis, W. L Ott. and N. M . Sine, "The Analysis of Nickel,' Pergamon Press Inc., London, 1966, p 160.
882
A N A L Y T I C A L C H E M I S T R Y , V O L . 46,
NO. 7,
J U N E 1974
Elemental copper and copper(I) chloride are dissolved according to these reactions. CuCl,
+ CUO
2CuC1
+ 2KC1
CuCl
=
2CuCl.c
= 2KCuC1,
+ HC1 =
HCuCl,
(3) C. L. Luke, Anal. Chem., 29, 1227 (1957). Marczenko and L. Chotuj-Lenarczak. Chem. Anal. (Warsaw), 10, 729 (1965). ( 5 ) I . M . Kolthoff and P. J. Elving. "Treatise on Analytical Chemistry," Interscience Publishers, New York, N.Y., 1961, Part I I , Vol. 7, pp 75, 81, and 85. (6) K . Meineke, Z. Anal. Chem., 10, 280 (1871); J. Chem. SOC. (Lond o n ) . 25, 89 (1872). (4) Z
