Spectrophotometric Estimation of Pentachlorophenol in Tissues and Water WILHELM DEICHMANN AND LAWRENCE J. SCHAFER Kettering Laboratory of Applied Physiology, University of Cincinnati, Cincinnati, Ohio
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ing 10 ml. of 4 per cent sodium hydroxide. The distillate is heated over a flame to about 75" C., and then half of this volume is measured into a 1-liter beaker, slowly concentrated on a hot plate to 50 ml., then transferred to a 250-ml. Erlenmeyer and evaporated to about 2 ml. [The other 500 ml. are set aside as a reserve to supply a suitable aliquot, in case the results of the first determination fall beyond the curve (Figure 2).]
ENTACHLOROPHENOL is used in industry as a n agent for the preservation of wood and wood products. Attention was first called t o its toxicity in studies on mice by Bechhold and Ehrlich, in 1906 (1). Extensive toxicity studies were carried out by Kehoe, Deichmann, and Kitzmiller in 1939 (S),and by Boyd, McGavack, Terranova, and Piccione in 1940 (2) and 1941 (4). Extension of the investigation necessitated the development of a n analytical method. The procedure reported here utilizes the formation and the spectrophotometric determination of a reddish-yellow pigment formed by the action of fuming nitric acid on pentachlorophenol. The method was found dependable, and of sufficient accuracy to enable the authors to follow this compound through the body and to detect amounts below the harmful levels. Further toxicological studies of this compound will be reported elsewhere.
Method The following method is suggested for the determination of from 0 t o 9 mg. of pentachlorophenol in 10 grams of biological material (except pure fat) and of from 0 to 50 mg. of pentachlorophenol in about 5 d.of water.
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Y I LLlGRAUS OF PENTACHLOROPHENOL
BETWEEN DENSITYAND CONFIGURE2. RELATIONSHIP CENTRATION, EMPLOYING 10-MM. CELLS
REAGENTS REQUIRED.Concentrated hydrochloric acid, specific gravity 1.19, 4 per cent sodium hydroxide] fuming nitric acid, and chloroform. ORGANS AND TISSUES.The sample is macerated in a meat grinder or mortar and, if necessary, mixed in a "Powermaster kitchen mixer". Ten grams are then weighed into the Pyrex distilling flask (Figure 1) and 20 ml. of water and 2 ml. of concentrated hydrochloric acid are added. The mixture is distilled until 1000 ml. of distillate have been collected in a flask contain-
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Four milliliters of fuming nitric acid are then slowly added and the preci itate clinging to the bottom of the Erlenmeyer is freed completefy with a stirring rod, which is then rinsed with 1 ml. of fuming nitric acid. The solution is placed in ice for 20 minutes and is then washed with about 100 ml. of water into a 250-ml. separatory funnel. The reddish-yellow pigment is extracted with three 8-ml. portions of chloroform, permitting 5 minutes for each separation. The combined chloroform extracts are washed with a single 100-ml. sample of water (to remove water-soluble nitrates) permitting 10 minutes for the separation, then filtered through 2 layers of Whatman No. 1, 9-em. filter paper wetted with chloroform into a 25-ml. glass-stoppered cylinder. After diluting with chloroform to 25 ml., the concentration 1s determined by means of standard curves (Figures 2 and 3) using 10mm. or 4-inch matched Aminco cells and the spectrophotometer at 460 mp.
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M C R a M Of PENTAWLORFWnrP
FIGVRE1
BETWEEN DENSITY AND CONFIGURE 3. RELATIONSHIP CENTRATION EMPLOYING INCH CELLS
DISTILLING APPARATUS 310
April 15, 1942
ANALYTICAL EDITION
OF CONTROL BLOOD, URINE, ORGANS, AND TABLE I. ANALYSES TISSUESASSUMEDTO BE FREEOF PENTACHLOROPHENOL
PentachloroSample
Pentachloro-
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%unodl
Sample
MQ. Rabbit blood, 10 ml.
0
0 0 0.02 0 0
75 ml.
0
0 0.01
100 ml.
0.01 0 0 0 0.01 0
Rabbit urine, 10 ml.
MU. Ground rat, 10 grams
0
Beef brain, 10 grama
0.03 0 0
0
Rabbit muscle, 10 grama Rabbit liver, 10 grams Rabbit heart, 10 grams Rabbit kidneys. 10 grams Human urine, 100 ml.
0 0 0 0.03 0.01 0.01 0.02 0
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Table I gives the values obtained by analyzing samples of normal blood and tissues for pentachlorophenol. Table I1 indicates the recoveries of pentachlorophenol added to blood, urine, and tissues, respectively. WATER. The steam-distillation is omitted in the absence of other material reacting with nitric acid. A 2-5 d.sample is pipetted into a 50-ml. Pyrex test tube, and from 3 to 15 ml. of fuming nitric acid are added (depending on the amount of pentachlorophenol in the sample); the whole is then allowed to stand in an ice bath for 20 minutes. From this point the analysis is continued as described above, 10-mm. cells being employed for the spectrophotometric, analysis. 1
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Figure 4 gives the transmission curve of the pigment formed under these conditions; Figures 2 and 3 give the relationship between density and concentration of pentachlorophenol. BLOODAND URINE. Whole blood or urine (10-ml. samples) is distilled directly, after treatment with 20 ml. of water and 2 ml. of hydrochloric acid, but only 300 ml. of distillate are collected in an Erlenmeyer containing 3 d. of 4 per cent sodium hydroxide. This is evaporated on a hot plate to 20 ml., transferred to a 250ml. Erlenmeyer, and evaporated to 2 ml. The analysis is then continued as under “Organs and Tissues”. In the case of blood, paraffin is added to reduce foaming.
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W A V E LENGTM (mp 1
FIGURE4. TRANSMISSION CURVEFOR PIGMENT FORMED
TABLEXI. RECOVERY OF PEXTACHLOROPHENOL (Added to 10-ml. samples of blood and urine and to 10-gram samples of organs and tissues) Added Recovered Added Recover ed
Mo
MO.
.
MU.
Mg.
0.14 0.14 0.145 2.45 4.54 4.54 6.8 6.8 6.8
0.12 0.12 0.125 2.50 4.40 4.31 7.0 7.4 6.7
Rabbit Blood
,---
0.03 0.03 0.03 0.06 0.06 0.06 0.11 0.11 0.11
Rabbit Urine (25-ml. samples) 0.08 0.09 0.09 0.10 0.18 0.18
0.09 0.09 0.10 0.08 0.19 0.19
Ground Rat 0.24 0.24 0.24 0.24 0.43 0.43 1.5 1.5 2.5 2.5 4.8
0.22 0.21 0.22 0.23 0.44 0.43 1.4 1.4 2.5 2.3 4.5
Rabbit Lung 2.3 5.3 5.1
2.5 4.8 4.8
Rabbit Fat
Human Urine 4.54 4.54 5.5 5.5 6.0 6.8 6.8 9.95
4.54 4.90 5.0 5.1 6.0 5.4 6.9 9.-6
Rabbit Muscle 2.5 5.0
2.4 4.9
Rabbit Liver 5.0 8.0 8.0
4.4 8.0 7.6
Rabbit Heart 2.5 4.5
2.55 4.0
Rabbit Kidneys 2.5 4.5
2.4 4.6
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Milligrom of Sodium Pentochlwphenote
FIGURE5 .
RELATIONSHIP BETWEEX CONCENTRATION
D E N s r r Y AND
10-nim. cells employed. Concentration of codium pentachloropbenate in sample of water is read directly from this graph.
Figure 5 gives the relationship between density and the quantity of pentachlorophenol (in milligrams) present in the original sample. The final estimation can be carried out with a colorimeter by using standard solutions. When 28 mg. of pentachlorophenol as the sodium salt were added to 2-ml. samples of water, the following quantities were recovered : 27.2, 27.4, 28.0, 27.7, 28.0, 26.6, 26.9, 27.7, 28.6, 28.6, and 29.1 mg. It is advisable to read the solutions in the PRECAUTIONS. spectrophotometer within 10 minutes; the color is stable, but droplets of water may settle out if the solutions are allowed to stand for longer periods.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Figures 2, 3, and 5 were obtained with the author’s photoelectric spectrophotometer and with their equipment; therefore, if accuracy is desired, it is not advisable to use them with other spectrophotometers and cells. It is suggested that other investigators prepare their own figures. The cells, even though they are matched, should not be interchanged and should always be placed in the same position. It is imperative that any precipitate which may form be loosened completely from the bottom of the Erlenmeyer after the 4 ml. of nitric acid have been added. Errors will not be introduced if samples go to dryness on the hot plate; the precipitate is brought in solution with about 2 ml. of water.
Discussion and Summary A method for the determination of pentachlorophenol in biological material and in water is based on the formation and
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spectrophotometric determination of a reddish-yellow pigment formed by the action of fuming nitric acid on pentachlorophenol. Substances other than pentachlorophenol may react with nitric acid to form colors which may occasionally give false positive results. I n no instance, however, did these errors exceed 0.03 mg. per sample (Table I).
Literature Cited (1) Bechhold, H., and Ehrlich, P., 2. physiol. Chem., 47, 173 (1906). (2) Boyd, L. J., McGavack, T . H., Terranova, R.. and Piccione, F. V., N . Y . Med. CoZZ. and Flower Hoap., Bull. 3, 323 (1940).
(3) Kehoe, R. A., Deichmann, W., and Kitsmiller, K . V., J. Ind Hvo. Tozicol.. 21. 160 (1939). (4) McGavclok, T. H.,‘Boyd, L. J., Piccione, F. V., and Terranova, R., Ibid., 23, 239 (1941).
Determination of Nitrites Discussion of the Shinn Method As Applied to Examination of Water N. F. KERSHAW, Indianapolis Water Company, Indianapolis Ind. AND N. S. CHAMBERLIN, Wallace 81 Tiernan Co., Inc., Rfontclair, N. J.
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HE diazotization of sulfanilic acid by the available nitrite in water, using a-naphthylamine as a coupling agent, has continued throughout the years as a colorimetric method for determining nitrites in water (1). The diazo reaction has been used for the determination of minute quantities of certain organic substances, many of which in turn could have been used for determining nitrites. One of the compounds diazotized and determined colorimetrically by available nitrite has been sulfanilamide. The diazotization of sulfanilamide by available nitrite, using a-naphthylamine as a coupling agent, was eliminated some time ago. Dimethyl-a-naphthylamine was next used, but found unsatisfactory because of the necessity of using a catalyst for rapid development of color in dilute solutions. Bratton and Marshall (3) investigated many new coupling agents for the diazotization of sulfanilamide and found .V-( 1naphthyl)-ethylenediamine dihydrochloride the most satisfactory. They state that this new coupling agent is far better than dimethyl-a-naphthylamine, because of its producibility and purity, greater rapidity of coupling, increased sensitivity, and increased acid solubility of the azo dye formed. Shinn (6) was the first to make use of sulfanilamide and the coupling agent, N-( 1-naphthyl)-ethylenediamine dihydrochloride, as a means of determining nitrites; in fact, her article was brought to the authors’ attention because she had found these reagents far superior to sulfanilic acid and a-naphthylamine. Shinn points out that sulfanilamide is more stable than sulfanilic acid, both in the dry state and in solution; furthermore, it reacts more rapidly in the coupling process. It has been recognized that the coupling of diazotized sulfanilic acid with a-naphthylamine is relatively slow (2) and that the color developed must be read within 30 minutes ( I ) because it is so unstable. The authors’ experience has shown that, with the standard procedure ( I ) , full color development is not reached until 30 minutes’ standing with subsequent fading after 30 minutes. Because of the qualification of the new coupling agent, the use of sulfanilamide, and the resulting improved diazo reaction, it appeared that the use of these two compounds for
determining nitrites in water would be worthy of consideration. The Shinn method (5) for nitrites was investigated in order to determine its applicability to water because of the need for a more satisfactory method than the standard procedure (1). The Shinn method, as published, prescribes a n unknown of no greater volume than 35 ml. which, after the addition of reagents, is diluted to 50 ml. This procedure would be awkward and contrary to best waterworks analytical procedure. Good analytical technique would necessitate the use of 50-ml. or 100-ml. water samples of unknown nitrite content since comparatively few waterworks laboratories have photoelectric colorimeters, but will continue to use for some time to come the Kessler tubes to which they are accustomed. The method emphasizes the increase in sensitivity with decreasing final volumes of the unknown nitrite solution. As published, the article points out that samples of unknown nitrite content, containing less than 0.0025 mg. of nitrite (NO2) or 0.000761 mg. of nitrite nitrogen (K)per 50 ml., or 0.050 p. p. m. of nitrite or 0.015 p. p. m. of nitrite nitrogen, gave colors too faint to be read with any reasonable degree of accuracy with a photoelectric colorimeter. If this were the case it would be impossible to apply the method to waterworks practice, since a large portion of the water supplies contain less than 0.015 p. p. m. of nitrite nitrogen. Any method to be given serious consideration must be sensitive t o 0.001 p. p. m. of nitrite nitrogen. Although Shinn recommended that the unknown be limited to 35-ml. volume, it was found by preliminary investigation that like amounts of nitrite nitrogen, treated before dilution, developed less color than when they were diluted to n final volume of 50 nil. before addition of the reagents. The sensitivity of the reagents increased with increasing dilution, and maximum color was not developed until the volume before the addition of the reagents became 40 ml. or more. This fact overcame the first objection to the method. This led to a n intensive study to determine how equivalent amounts of nitrite nitrogen, developed in dilute or concentrated volumes, would give equivalent readings for all amounts
