sorption level. A very close agreement was found between the standard deviations of the measured blank readings a t the respective lead and bismuth wavelengths and the standard deviations calculated from the corresponGing fluctuational concentration limits, defined by RamlrezMuiioz ( 3 ) as the peak-to-peak noise at zero absorption level, and set equal to 4 n. Taking a 95% probability, one
can expect the spread of results for 2 CT on both sides of the true value. Due to instrumental fluctuations close to zero concentration, the uncertainty range which determines the lower concentration limit has been found to be 1 pg lead, and 2 pg bismuth, respectively.
Rami&-Muiioz, "Atomic Absorption Spectroscopy," Elsevier Publishing Co..Amsterdam-New York, 1968, p 227.
Received for review October 2, 1972. Accepted March 2, 1973.
(3) J.
Determination of Thiocyanate as a Rhodamine B Complex Ariel H. Guerrero and Antonio M. Roig facultad d e Ciencias Exactas y Naturales, Ciudad Universitaria, Nhiez, Pabellon 2, Buenos Aires, Argentina
The determination of small quantities of thiocyanate is commonly done through the reaction with iron(II1) in acid media ( I ) . This method has several drawbacks, the main ones being moderate stability and poor sensitivity (5 to 10 pg/ml). In another method used for biological fluids ( Z ) , the sample is treated with bromine in order to convert thiocyanate into cyanide bromide, which after elimination of excess reagent with As3+, reacts with benzidine and pyridine solution. Cyanide interferes, but sensitivity is good (1 crg/ml). We have previously proposed (3) the reaction between thiocyanate and rhodamine B in benzene for the determination of small quantities of this ion ( 4 ) . In this method, after extracting an acid solution of thiocyanate with benzene and adding colorless rhodamine B solution dissolved in benzene to the isolated organic phase, a purple color is obtained, similar to the reagent aqueous solution. In this paper, we wish to report that the method may be considerably improved if thiocyanic acid is extracted in the presence of sulfite ion, salting out with sodium sulfate. The reaction proceeds efficiently if water is eliminated from the organic phase. Stability and sensitivity are satisfactory. EXPERIMENTAL Apparatus. A Bausch & Lomb Spectronic 20 was used for all absorbance measurements. Reagents. AR grade reagents were prepared as follows: standard thiocyanate solution (25 pg/ml as SCN-); rhodamine B solution (0.01M in benzene. Drug is purified according to ( 4 ) by dissolving in absolute ethanol and precipitating with 10 volumes of ethyl ether.); sodium sulfate (saturated solution); sodium sulfite (saturated solution); and sulfuric acid (30% (w/w), saturated with sodium sulfate). Procedure. Place 0.05 ml of sample in a 5-ml centrifuge tube. A small drop (ca. 0.02 ml) of sodium sulfite, 1 ml of benzene, and two drops of the sulfuric acid solution are added successively. Cover, shake for 1 min, and centrifuge for 2 min a t 2500 rpm. Transfer the organic phase carefully with a dropper, without touching the aqueous solution, to a dry tube (5 ml) and add 0.5 P. Dansen, H. E. Bass. 8. Dandou, and E. W. Jones, Arch. Eur. Health, 14, 865 (1967). W. N. Aldridge, Analyst (London), 70, 474 (1945). A. M . Roig and A. H. Guerrero, "Determination of thiocyanate and iodide by means of rhodamine," XX IUPAC Congress, Moscow, 1965. A. M .
Roig. Ph.D. Thesis, Facultad de Ciencias Exactas y Naturales, Univ. de Buenos Aires, 1963.
ml of 0.01M rhodamine B solution in benzene. A purple color is a positive signal, the sensitivity limit of identification being 0.5 pg and of concentration, lO-5M. If the sample is obtained from an alkaline treatment, it should be neutralized with 30% sulfuric acid. Under these conditions, there are no interferences from common ions; b u t if they are present, SeCN-, TeCN-, and W2would give a positive reaction ( 5 ) . On the other hand, it is well known that SbCls- and SbI4- react with rhodamine B under conditions similar to those necessary for thiocyanate to react and we have found that BiI4 may be specifically extracted and gives a positive signal (6). Iodide ion forms a complex in water solution which, extracted with benzene, is also purple ( 3 ) . The quantitative technique coincides with the above description, b u t the volumes are changed. Sample, 1.0 ml, is placed in a 25-ml centrifuge tube and 1 drop of sulfite solution, 10.0 ml of benzene, and 2.0 ml of the sulfuric acid solution saturated with sodium sulfate, are added successively. Cover, shake vigorously for 1 min, and centrifuge a t 2500 rpm for 2 min. Transfer, with the same precautions as mentioned above, 5.0 ml of the benzenic extract to a dry tube, to which 5.0 ml of the reagent solution are added, and after mixing, covering, and waiting 1 min, absorbancy is read a t 560 nm and 20 "C.
RESULTS AND DISCUSSION The absorption spectrum of the complex was read from a 20 pg/ml SCN- solution. It is very similar to the one obtained with the other ions which react, and to that of pure rhodamine in water (peak a t 560 nm). Beer's law is followed between 5 pg and 20 Fg, the limit for the instrument being 25 pg. The influence of temperature on rhodamine reactions is known and to choose an optimum (20 "C), trials were run with the results shown in Table I. The concentration of the reagent in benzene is not critical between 0.01M and O.O02M, but by working with a concentration of 0.01M better results are obtained. F o r instance, the absorbance of a 20 pg/ml SCY- solution gives the range shown in Table 11. Precision was studied while working under the conditions chosen for the technique proposed in this paper (Table ID). Extraction of HSCN is favored by salting out with Nad304 in the 30% HzS04 solution, with an estimated efficiency of around 95% (Table IV). When applied to saliva and urine with known added ( 5 ) A. H. Guerrero, A. M . Roig, and C. R u i z , Sesiones Quimicas Argentinas,,San Luis 1970. (6) R . Sivori and A. H. Guerrero, A n . Asoc. Quim. Argent.. 55, 157 (1967),
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
1943
Table I. Absorbance of Rhodamine 6 Thiocyanate in Benzene Solution-Influence of Temperature SCN-/ml
10 PS
15 Pg
20 Pg
20 "c 25 "C 30 "C
0.320 0.295 0.230
0.480 0.430 0.375
0.720 0.660 0.540
Table II. Absorbance of Rhodamine B Thiocyanate in Benzene Solution-Influence of Reagent Concentration Reagent concn in benzene
0.01 M
0.005M
0.002M
0.001M
A
0.720
0.710
0.700
0.650
Table I l l . Precision of the Quantitative Reaction for Thiocyanate with Rhodamine B (Absorbance) SCN-/ml
1 2 3 4 5 6 7 X
S
s%
5
wcs
0.170 0.180 0.175 0.165 0.180 0.170 0.165 0.172 0.0064 3.7%
10 Pg
15 Pg
20 Pg
25 k g
0.320 0.340 0.300 0.310 0.330 0.330 0.31 0 0.320 0.014 4.3%
0.480 0.470 0.470 0.480 0.480 0.470 0.500 0.480 0.01 1 2.3%
0.720 0.700 0.710 0.700 0.730 0.730 0.740 0.720 0.0125 1.7%
0.840 0.810
n (number of samDles in each level) ?, = averaae value; s% = s/T.100.
=
0.870
0.830 0.840 0.860 0.810 0.837 0.023 2.7%
7: s = standard deviation;
Table IV. Absorbance of Rhodamine B Thiocyanate in Benzene. HSCN Extracted with Different Concentrations of Na2S0.1in 30% H2S04 5
10
15
20
25
30% H2S04 30% H2S04
0.030
0.075
0.105
0.130
0.180
Na2S04
0.145
0.260
0.370
0.560
0.715
fig SCN - / m l
+ 15% 30% H2S04 + 35% Na2S04(satd)
0.170
0.320
0.480
0.700
0.840
quantities of KSCN, recovery was 92 and 95%, respectively with precision similar to that of pure solutions. For saliva samples, the per cent standard deviation (sW)is 4.2 to 3.2%, and for urine, s% is 4.0 to 2.8% over the range 5 to 20 pg/ml. This reaction was discovered by us ( 4 ) when testing pseudo-halogenic ions as interferences for the antimony(II1) reacting as iodide (SbI4-), after extraction in benzene, with rhodamine B (7). This reaction and the classical one of this same reagent with antimony( V) have been explained through RHSb14 and RHSbC16 formation, where R represents rhodamine B (8). Our working hypothesis is that various acids, H,X are extracted in benzene and those which are strong enough react with rhodamine B, forming ion association complexes with the general formula (RH+),Xn-. The anions may or may not be oxo complexes and for reactions known for a long time, as with Au(III), we suggest RH+MCl*- as a general formula of the colored compound. (7) P West and W C Hamilton Anal Chem 24, 1025 (1952) (8) C Kutnetzov J Anal Chem Russ 2. 179 (1947)
1944
The anions which up to now we have tried in benzene with positive results (5) are as follows: SCN-, Sb14-, SbCle-, Bi14-, SeCN-, TeCN-, and N3-, the two antimony anion reactions being known beforehand. Salicylic acid reacts, but not benzoic acid or anhydrous acetic acid dissolved in benzene. Hydriodic acid is not extracted from water solutions because of high dissociation, but if rhodamine B is added, purple RH+I- is extracted in benzene with low sensitivity, the identification limit being 40 pg. In a recent paper (9),purified rhodamine B molybdophosphate is extracted with chloroform-butanol and determined by means of a spectrofluorimetric method. The authors suggest (R+)3PMo3- as the formula for the complex, but it seems preferable to use the formula (RH+)3PMo3-, with the same meaning but with the proton written explicitly. Presumably, the other heteropolyacids are also extractable, forming compounds of rhodamine B with a similar formula. Further work is in progress to confirm and develop these results because, for instance, Sb13 has been suggested as a possible agent for the color development in benzene (IO), while our hypothesis requires the reaction of HSb14, or a t least the formation of HI in the organic solvent. The spectra of various rhodamine B salts in benzene have the peak ca. 560 nm and they are very similar to the spectrum of the aqueous solution of the reagent (11). This is a strong argument for the protonized cation rhodamine B in benzene solution as the colored resonating species with an undissociated carboxyl. In brief, our working hypothesis is as follows: a ) an acid H,X, X n - being any adequate anion, is extracted in benzene solution; b) rhodamine B in benzene solution (colorless) is added; c) the acid, if strong enough, protonizes the reagent and its colored cation forms an ion association complex of the type (RH+),X,-. This complex is also formed in water solution by the above-mentioned acids and may be extracted in benzene. Even some very strong acids such as hydriodic, which because of dissociation in water is not extractable in benzene, form the complex with rhodamine B in water and are soluble in the organic phase as such.
CONCLUSIONS Thiocyanate may be determined by means of rhodamine B in benzene solution, following acid salted-out extraction. Sensitivity is better than usual for reactions of thiocyanate, and selectivity is acceptable considering that most interfering ions are not common or may be avoided by a prior alkaline treatment. An explanation is suggested based on forming colored ion association complexes of rhodamine B with acids strong enough to protonize it. The West and Hamilton reaction for Sb(II1) is a particular case which can be explained by these same arguments. Several other references confirm the reliability of the suggested hypothesis (12-15). Controversial ones ( I O ) are being studied experimentally. Received for review October 18, 1972. Accepted February 22, 1973. (9) G. F. Kirkbright, P. Narayanaswamy, and T. S. West, Anal. Chem., 43, 1434 (1971). (10) R. W. Ramette.Anal. Chem., 30, 1158 (1958). (11) R. W. Ramette and E. Sandell. J. Amer. Chem. SOC.. 78, 4872 (1956). (12) G. Martin, Buli. SOC.Chim. Biol., 34, 174 (1952). (13) 6.Tsvaroha and 0. Mala, Mikrochim. Acta., 1962, 634. (14) 2. Slovak and M . Pribyl, Collect. Czech. Chem. Commun. 31, 1742 (1966). (15) V. Meketroz and J. Kahlicek. Fresenius' 2. Anal. Chem.. 208, 7 (1965).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
