Colorimetric Determination of Microgram Quantities of Sodium and Copper Pentachlorophenates W. T. HASKINS National Microbiological Institutes, National Institutes of Health, Bethesda, Md.
This study was undertaken to develop a simple, rapid method of determining microgram quantities of sodium and copper pentachlorophenates in dilute aqueous solutions, chiefly as an aid in the study of their molluscacidal properties in natural waters. Wallin’s method for estimating pentachlorophenates was modified in order to eliminate its objectionable blank and adapted to read the results with color standards for field use. It is capable of estimating concentrations between 1 and 100 p.p.m., using a 5-ml. sample. A more precise spectrophoto-
S
ODIUM and copper pentachlorophenates have shown considerable promise aa molluscacides for the destruction of snail intermediate hosts of the human schistosomes (1, 2’). These snails are found in aquatic environments such as streams, irrigation ditches, swamps, and lakes. I n field trials of the compounds in such natural waters, it is desirable to know the actual cmcentration prevailing at various locations throughout the body of water, in order that it may be correlated with other pertinent data To be of optimum value the analytical method should be capable of use in the field as well as in the laboratory and hence should involve a minimum of equipment to facilitate portability. As these compounds are effective a t concentrations of 10 p.p.m. or less, the method should also be sensitive enough to detect 1 p.p.m. and versatile enough to include a range up to 100 p.p.m. without requiring extensive serial dilutions of the sample. KO published method of analysis for these compounds met thew criteria. Wallin (9) has reported that methylene blue combines quantita; tively Tith sodium pentachlorophenate a t a pH of 10.9 to form a blue-colored complex which is soluble in chloroform. However, he found that the use of this property as a basis for a colorimetric determination was complicated by a magenta-colored blank obtained when the alkaline methylene blue solution was extracted with chloroform. Preliminary tests indicated that this method was sufficiently sensitive to meet the desired requirements, provided it could be simplified by using color standards instead of a spectrophotometer to read the tests. The chief source of difficulty was the magentacolored blank which completely obscured the blue of the complex in the low concentration range. It was found that this interference could be eliminated by mixing equal proportions of an aqueous solution of methylene blue chloride and saturated sodium bicarbonate and extracting the mixture with chloroform until the magenta color was removed. The bicarbonate-methylene blue solution could then be used as a reagent for the test for periods up to 1 week without interference from chloroform-extracted color. The pH of the reagent is approximately 8.5. This is sufficiently alkaline for the quantitative formation of the methylene bluepentachlorophenate complex without further adjustment, thus eliminating the need for any additional buffers in carrying out the test. REAGENTS REQUIRED
Methylene Blue Chloride Solution. A 0.02’3Z0 solution is prepared from certified dye (CI 922). The weight of material taken for the preparation of this solution is corrected for the actual dye
metric method was developed for use in the laboratory based on the formation of a chloroform-soluble complex between tetra- and pentachlorophenates and safranin-0 in bicarbonate-buffered solutions. The latter is useful in determining 5 to 50 micrograms in a S m l . sample. With suitable modifications, such as extraction or steam distillation techniques, both methods may be adapted to the determination of pentachlorophenols in solids or nonaqueous solutions requiring relatively small samples as compared to older methods of analysis.
content of the dye lot, as stated on the label, so that 100 ml. of solution contain 20 mg. of actual dye. Bicarbonate-Methylene Blue Reagent. A mixture of 1volume of 0.02% methylene blue chloride solution and volume,of saturated sodium bicarbonate solution is extracted with successive 1-volume portions of chloroform until the chloroform extract is colorless or nearly so. Usually four or five extractions are sufficient. (The chloroform used for the extractions may be recovered by shaking it with an equal volume of dilute hydrochloric acid, then by washing twice with equal volumes of water. The recovered solvent may be used for preparation of the reagent, but not in the test procedure.) The aqueous phase is stored in a tightly closed bottle with a minimum of exposure to strong light. It remaine usable for about 1 week or until 1 ml. of it diluted to 5 ml. with water and shaken with 5 ml. of chloroform develops an appreciable pink color in the chloroform layer within a 15-minute period.
Table I.
Dilution Factors for Preparation of Color Standards
5 - d . sample, p.p.m. 1 rnl. of 0.02% methylene blue chloride solution diluted to, ml.
2
5
10
25
50
100
60
30
20
10
5
3
Color Standards. The color standards are prepared bv suitable dilution of the 0.02% methylene blue chloride solution. The values for these dilutions were determined by matching the colors produced by known concentrations of pure sodium pentachlorophenate with the proper dilution of the dye solution. If water is used as the diluent, the life of the standards is short as judged by fading and the deposition of insoluble material. The life of the standards may be increased to 2 to 3 weeks if 0.1 N hydrochloric acid is used as the diluent. Table I gives the dilution factors for the preparation of the standards. The figures on parts per million are based on the use of a 5-ml. sample for the test. Approximately 10 ml. of each standard are placed in a screw-capped 16 X 150 mm. culture tube and protected from strong light when not in use. The same standards may beused for the determination of copper pentachlorophenate solutions, aa the pentachlorophenol content differs from the sodium salt by only approximately 3%, which is not detectable by this method. (The culture tubes as supplied by the Kimble Glass Co. have a coated paper liner over a cork backing in the cap. This coating is soluble in chloroform and should be removed and replaced with an aluminum-foil disk in the caps on the tubes used for the test. The coated paper liners should be retained for the caps used for the tubes containing the acidic color standards.)
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V O L U M E 2 3 , NO, 11, N O V E M B E R 1 9 5 1 Reproducibility of Color Standards. I n order to test the reproducibility of the color standards with varying sources of methylene blue chloride, five different lots of the certified dye from various suppliers were obtained. The stock 0.02% solution of each was prepared and dilutions were made for the standards as given in Table I. When inspected in the 16-mm. tubes, agreement among the different lots was excellent. The bicarbonatemethylene blue reagent was also prepared from each of the stock solutions and tests were made a t 2 and 10 p.p.m. on sodium pentachlorophenate. The colors produced in the tests matched very well Rith the corresponding color standards. Thus it may be concluded that no great variation in results may be expected with different lots of certified dye. TEST PROCEDURE
Place 5 ml. of the water to be tested in a 16 X I50 mm. screwcapped culture tube and add 1 ml. of the bicarbonate-methylene hlue reagent and 5 ml. of chloroform. Close the tube tightly and shake it vigorously for 15 seconds. Place the tube upright, and as soon as separation of the layers is complete inspect the upper layer. If it is definitely blue the sample contains 10 p,p,m. o r less o f pentachlorophenate, in which case the color in the lower layer is compared with the standards to obtain the concentration. (Comparison of the color of the chloroform layer in the tubes with the color standards is greatly facilitated by use of a simple wooden comparator block having three holes into mrhich the tubes can be slipped. Transverse slots are cut through the block near the bottom, so that only the chloroform layer is visible when the tubes are in place. A piece of ground glass cemented over one end of the slots will provide more even illumination and freedom from troublesome reflections.) With concentrations greater than 10 p.p.m., the upper layer mill be colorless or a very pale blue after shaking with 1 ml. of the reagent. I n this case add 1 ml. more of the reagent and shake the tube as before. Repeat until a definite blue color is present in the upper layer. Then compare the color of the lower layer with the standards to estimate the concentration. I n general it will require an additional 1 nil. of reagent for samples containing 25 p.p.m., 2 ml. for 50 p.p.ni., and 4 ml. for 100 p.p.m. before a definite blue color is observed in the upper phase. A moderate excess of reagent does no harm. After extraction of the color into the chloroform, the tests should be read as soon as the chloroform layer is clear (3 to 5 minutes) and in any case within 30 minutes. This limit is necessary because a pink color develops in time in the chloroform, which makes comparison n-ith the standards expecially difficult in the range of 1 to 5 p,p.m. It is advisable to run a blank determination on 5 ml. of the water from the stream before treatment with the pentachlorophenate in order to eliniinate the possibility of interfering substances. A blank is also useful in estimating concentrations of the order of 1p.p.m. where the color will be lighter than the 2 p.p.m. standard but definitely bluer than the blank. The practical accuracy of the method is about 20% error in the region of 5 to 100 p.p.m. and &I p.p,m. belom- 5 p . p n REPRODUCIBILITY OF T E S T
The sodium and copper pentachlorophenates used as molluscacides are technical grade materials and are subject to variation in pentachlorophenol content and the kind and amount of impurities present. Thus different lots of the chemicals might be expected to give varying results on observed values for the concentration of solutions prepared from them on a purely weight basis. Samples of commercial sodium pentachlorophenate, Dowicide G (Dow Chemical Co.), Santobrite pellets and Santobrite briquets (llonsanto Chemical Co.), were obtained and solutions of knoiln concentration by weight were prepared from them. Upon analysis of these solutions by the test procedure, it was found that no visible difference in the intensity of the color was produced among the various samples for comparable concentrations and that they also matched the proper color standards. The color standards were originally standardized against known concentrations of sodium pentachlorophenate prepared from pure pentachlorophenol. This result seemed remarkable, considering that the technical samples were labeled as containing from 74 to 79y0 sodium pentachlorophenate, 11% other sodium chlorophenates, and 10 to 15% inert matter. It was apparent that some of the other chlorophenates must also be forming a chloroform-soluble
1673 caomplex with the methylene blue; otheraise the tests would be noticeably lighter than the standards. The sodium salts of phenol, 2,4dichlorophenol, 2,4,5- and 2,4,Btrichlorophenol, and 2,3,4,6tetrachlorophenolwere examined for their ability to form the complex under the test conditions; only the tetrachloro compound did so. It may then be concluded that technical sodium pentachlorophenate contains sufficient tetrachlorophenate to give results with this test which are comparable to those given by pure sodium pentachlorophenate within the limits of error of the method. The test seems to be specific for the tetra- and pentachloro derivatives to the exclusion of the lower chlorinated memhrs. Only two samples of technical copper pentachlorophenate (Monsanto Chemical Co.) were available for testing. Both gave good matches with each other and Rith the standards. INTERFERIVG SUBSTAYCES
Hard water, water containing appreciable amounts of iron, or copper pentachlorophenate will produce cloudy precipitates with the bicarbonate-methylene blue reagent. The precipitate is usually carried into the chloroform layer as a suspension and makes comparison Rith the standards difficult. This intei ference is readily eliminated by dissolving a few milligrams (four or five small crystals) of sodium citrate in the 5-ml. sample before adding the reagent. This prevents the metals from precipitating and does not otherwise affect the test. SPECTROPHOTOMETRIC METHOD
Where greater precision in mtasuring the concentrations ia desired, the above method may be adapted to read the density of the color produced by the complex nith a spectrophotometer. The method described below was, however, found to be superior for use with a spectrophotometer because it gave more consistent results and did not require the use of the rather unstable methylene blue-bicarbonate reagent. I t was found that safranin-0 (CI 841) also forms a chloroformsoluble complex with tetra- and pentachlorophenates in much the Fame way as methylene blue. Furthermore, extraction with chloroform of bicarbonate-buffered aqueous solutions of this dye gave only a faint pink color in the chloroform, thus eliminating the need for pretreatment of the reagent to prevent the formation of an undesirable blank This dye was not suitable for use nith color standards because the color of its complex with the pentachlorophenate in chloroform cannot be matched with standards prepared from it. This difference in the color of the complex is due to a greater absorption of the wave lengths in the region of 530 to 550 mp than is present in aqueous safranin-0 solutions. S o simple way of preparing stable standards was found. Reagents Required. A 0.0274 aqueous solution of safranin-0 prepared from certified dye (CI 841), a saturated solution of sodium bicarbonate, and chloroform. ( I t is not necessary to correct for the actual dye content of the dye lot in preparing this solution, since it is not used as a color standard.) Procedure. A 5-ml. sample of an aqueous solution Containing 5 to 50 micrograms of the tetra- or pentachlorophenate is placed in a glass-stoppered or screw-capped 16 X I50 mm. tube, 5 ml. of chloroform are added and folloiTed by 0 5 ml. of saturated sodium bicarbonate solution and 0.5 ml. of 0.02% safranin-0 solution. The tube is closed and shaken vigorously for 15 seconds. After allouing about 5 minutes for the layers to separate and become clear, 3 to 4 ml. of the chloroform layer are removed from the tube with a capillary pipet and transferred to a 10-mm. cuvette which is loosely stoppered to prevent excessive evaporation. (The author used Corex cells and covers with a Beckman DU spectrophotometer.) A blank determination is run at the same time using 5 ml. of water and the same reagents. The optical density of the unknoivn is determined in the region of 520 to 550 mp after setting the instrument to zero with the blank. The concentration is then obtained from a calibration curve prepared from known concentrations of the tetra- or pentachlc rophenate in question. With each of the samples of these chemicals examined, the curves have been straight lines passing through the origin for
ANALYTICAL CHEMISTRY
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amounts up to 50 microgram. The density readings were 0.8 to 1.0 a t the upper limit. The curves for technical grades of sodium pentachlorophenate lie closer t o the curve for the pure material than would be expected, probably for the reason discussed for the methylene blue method. When 5-ml. samples of s n aqueous solution containing 25 micrograms each of technical sodium pentachloraphenate were analyzed, the following amounts were recovered: 23.6, 22.6, 24.0, 26.4, 22.4, 25.2, 24.2, and 27.0 micrograms. Precautions. The chloroform layer should be removed from the tubes within 30 minutes from the time of extraction because the color darkens on standing in contact with the aqueous phase; after removal the color remained stable for at least an hour, provided evaporation of the solvent was prevented. The water used
for the blank should be the same as that in the unknown solution; provision for this should be made when conducting field work and the samples are to he examined in the laboratory. Iuterfering ions, such as those found in hard water, and iron or copper may be dealt with by adding a small amount of sodium citrate to the sample as explained above undcr the methylene blue method. LITERATURE CITED
(1) Berry. E. G.. Nolan, M. O., and GonaBles, J. 0.. Pub. Health Rapt.. 65,939-50 (1950). (2) Nolan, M. 0.. and Berry, E. G.. Ihzd., €4,942-9 (1949). (3) Wallin, G. R..ANAL.CHEM..22, 1208-9 (1950). R E C E I VA ~ ~Dr i 4. l 1951
lsoquinoline in Chemical Microscopy HAROLD F. SCHAEFFER, Valparaiso University, Valparaiso, Ind. Although many heterocyclic nitrogen compounds, including quinoline, are usefill for the detection of various ions, no previous workers have reported on the miemchemical applications of isoquinoline. The investigation reported was carried out to determine the feasibility o f applying isoquinoline as a reagent in ohemioal microscopy. A procedure has been developed whereby isoquinolinium hydrochloride, in the presenoe of thiocyanate ion, can he employed for the microehemioal detection of zinc, cadmium, copper, and whalt, because each yields characteristic microcrystals with the reagent. One advantage of the prooedure is the ease with which cobalt and copper can he deteoted in the presence of each other and of nickel. Positive tests can be ohtained on a drop of solution, with the copper concentration as low as 1 part in 15,000 and the wbalt eoncentration as low as 1 part in 4000. For zinc the sensitivity approximates that of copper, but f o r oadmium it is only approximatelyone tenth as great.
A
LTHOUGH several ohemists (I*) have shown that quinoline m the presence of ammonium thiocyauste may serve m a reagent for the microchemical detection of certain cations, little attention has been given to the merits of isoquinoline and ammonium thiocyanate. Recently Spakowski and Freiser ( 4 )demonstrated that these reagents can he used for the quantitative precipitation of copper and zinc, hut they did not investigate the possibility of adapting the isoquinoline-rtmmonium thiocyanate combination to ohemical microscopy. It has now been found that the reagent oan be employed in the microchemical detection of several cations, because the resulting metallo-organic compounds separate in the form of characteristic microsoopic crystals.
PROCEDURE
In performing the test far vinc ion a small drop of the prepared reagent is caused to flow into a droplet of dilute test solution on a slide. In the presence of various aino salts, including the acetate, chloride, sulfate, and nitrate, characteristic crystals and clusters will separate (Figure 1). The composition of these crystals corresponds t o the formula Zn(CQH,N)x(CNS)2. With zinc acetate, for example, goad testa have been obtained on solutions containing 1 part of zinc in 15,000. Using a teat drop of 15 cu. mm. the reaction may detect the presence of 1microgram of Einc. The behavior of dilute solutions of ~ i n cchloride is approximately the same as that of the acetate, hut the presence of zinc nitrate appears to decreme the sensitivity. The temperature of the laboratory also influences the sensitivity of the tests, as very dilute solutions did not respond as well when the temperature was unusually high for a prolonged period. In the presence of cadmium acetate the isoquinoline-ammonium thiooyanate reagent causes the separation of characteristic orystals (Figure 2). Good tests have been ohtaiued with a oadmium ion concentration of 1 part per 1000, but at a dilution of 1 part in 5000 the test is not reliable. Because of the small dimensions of the crystals generally formed in the cadmium test, it is preferable to observe them through an &mm. objective instead of the more common 16-mm. lens. The test is much less
REAGENT
hydrochloride and 0.5 M-in reapeat t o thiocyanate. ‘As st this concentration part of the solute may eventually separate from solution, it is advisable to work with a frefih reagent prepared from two stable stock solutions. One of these is a 1 M solution of ammonium thioevrtnate. The other is a 0.4 M solution of iso~
~
0.2 .“m.
tiins.- The iioq$noline c&stks obtained by freezing the Eastman Xodek Ca. “practical” grade were found satisfsotory for the purpose.
Figure 1. Crystals Formed by Reaction of 1soquinolir.eAmmonium Thiocyanate Reagent with Zinc Chloride
