insignificant quantities of carbonyl impurity. Occasionally, samples will contain a relatively high carbonyl impurity. These samples are usually analyzed by diluting an appropriate weighed portion to a known volume with Solution I and pipetting a suitable aliquot. If an aliquot other than 5 ml. is chosen, a reference solution containing the same volume of Solution I as the aliquot must also be prepared before determining the carbonyl by the described procedure. An excess of up to 0.2 ml. of concentrated hydrochloric acid and double the usual 2,4dinitrophenylhydrazine concentration had no adverse effect on the formation of the 2,4dinitrophenylhydrazone; however, an undesirable feature of excess hydrochloric acid occurs when potassium hydroxide is added, because an excessive amount of potassium chloride forms and some of it precipitates. When this happens, the mixture can be filtered rapidly through paper to remove the precipitate and the absorbance determined in the usual manner. Water is added to the reaction mixture to ensure solution of the potassium
chloride formed from the addition of potassium hydroxide to the reaction mixture containing the proper concentration of hydrochloric acid. Additional water can be added, if desired, without affecting the results as long as a single-phase solution is maintained. Since the procedure described is a general one and utilizes the solvent properties of a hydrocarbon, only a limited quantity of additional water can be added. If 430 mp (Aax) were chosen for the work, the increased sensitivity would allow determination of only very low concentrations of carbonyl without modifying the procedure. The linear absorption at 480 mp, Figures 1A and 2 A , was chosen for the work, because the sensitivity allows the accurate and reproducible determination of a wide range of carbonyl concentrations. The standard curve should be established each time a new Solution I is prepared, because of the possible difference in carbonyl level between different batches of alcohol and hexane used. The most critical part of the procedure occurs immediately after the addition of potassium hydroxide. It is extremely important that the absorb-
ance of the wine-red 2,4-dinitrophenylhydrazone be determined within the prescribed time interval, because the absorbance decreases wtth respect to time. We have observed that, although the absorbance decrease is linear with respect to time for both aldehydes and ketones, the decrease for the hydrazones of aldehydes is much greater than that for aliphatic ketones after the initial 1.5minute interval. Within the prescribed 8- to Isminute reading interval, errors incurred will be small and within the accuracy of the procedure. LITERATURE CITED
(1) Jones, L. A., Hancock, C. IC. J . A m . Chenz. SOC.82, 105 (1960). (2) Jones, L. A,, Hancock, C. K., J . Org. Chem. 25, 226 (1960).
(3) Jones, L. A. Homes, J. C., Sebgman, R. B., ANAL. HEM. 28, 191 (1956). (4) Lappin, G. R., Clark, L. C., Ibid., 23, 541 (1951). (5) Lohman, F. H., Ibid., 30,972 (1958). (6) Mathewson, W., J . Am. Chem. SOC. 42, 1277 (1920). (7) Mendelowitz, A., Riley, J. P., Analyst 78, 704 (1953). (8) Toren, P. E., Heinrich, B. J., ANAL. CHEK 27, 1986 (1955).
RECEIVEDfor review May 10, 1963. .4ccepted September 30, 1963.
Separation of Hexachlorophene from Organic Matter Prior to Spectrophotometric Estimation V. D. JOHNSTON and P. J. PORCARO Analytical laboratory, Sindar Corp., New York,
b Hexachlorophene can b e isolated from a wide variety of natural substances, such as animal tissue, blood, milk, and peelings from fruits and vegetables, After a dehydrating and blending process, the extraction method involves destruction of the natural organic material with sulfuric acid. Hexachlorophene can then be determined by a spectrophotometric procedure which uses the 4-aminoantipyrine reaction. Examples are given of the successful recovery of hexachlorophene from biological specimens to which known amounts of hexachlorophene had been added.
H
U.S.P., 2,2’methylene bis (3,4,6-trichlorophenol), is being used in such a wide variety of applications that it has become not only a household term but also an analytical challenge as to its isolation and quantitstion. Separate monographs describing hexachlorophene and EXACHLOROPHENE
124
ANALYTICAL CHEMISTRY
N. Y.
hexachlorophene liquid soaps are listed in the U.S.P. (9) which includes an assay method by Childs and Parks (1). For some of the more common applications in toilet soaps, cosmetics and pharmaceuticals, hexachlorophene is determined by established procedures described in the manufacturer’s technical bulletin (IO), A modified colorimetric procedure of Gottlieb and Marsh (3) a titrametric, and two ultraviolet methods are discussed in this bulletin. A compilation of over 250 references to hexachlorophene includes very few analytical methods. None deals with its isolation from complex organic matter. Methods of assay for hexachlorophene involve either a solvent extraction from soaps and cosmetics, if necessary, or direct determination by a color producing reagent or by ultraviolet spectrophotometry. Lord, McAdams, and Jones (8) describe a direct ultraviolet method for use in soap, while Jungermann and Beck (5) first use dimethylformamide as an extraction
solvent in germicide preparations followed by ultraviolet determination. Clements and Newberger (2) use a separation based on a series of extractions from creams and pastes, the interferences being surface active agents. Methods are described for either modifying the n-ell known 4-aminoantipyrine reaction (4, 6) or using new color reagents (4,7). In many cases, components of the particular vehicle or other active ingredient in a soap, cosmetic cream or lotion, toothpaste, or drug will interfere with these tests and consequently a separation technique must be developed. Hexachlorophene in the presence of many organic interferences can be determined satisfactorily after employing a preliminary isolation procedure developed in our laboratory. Uses of hexachlorophene have become so diverse that a method for its reliable estimation is essential to guide the course of investigation. To illustrate, studies are
~
being made on the uc5e of hexachlorophene in agricultural sprays and veterinary medicine as an anthelmintic, thus requiring analytical separations from fruits and vegehbles, as well as milk, blood, and anim:tl tissue.
I
-
1
l
EXPERIMENTAL
A Waring high speed Blendor and a Beckinan DK2 spectrophotometer were used in this work. Reagents. All chemicals used are reagent grade. All solutions are aqueous unless otherwise specified. To prepare ammonia buffer, 3.88 grams ammonium chloride and 28.5 ml. of ammonium hydroxide (2801,) are diluted to 100 ml. Two milliliters of this solution is further diluted to 1000 ml. Preparation of Calibration Curve. Pipet appropriate aliquots of hexachlorophene dissolved in ethanol to contain 20 t o 100 p g . into a series of 2 j m1.-volumetric flasks. Add 0.5-ml. of freshly prepared 2% 4-aminoantipyrine solution to Each flask. The solutions are diluted t o mark with ammonia buffer solution, stoppered, and mixed. Then, 0.25 ml. of 8% potasqium ferricyanide solution is added t o earh flask. A blink is prepared .similarly. The absorbance is read 5 minutes after color development. The absorbance a t 475 mu us. concentration will yield a straight line plot. Figure 1 depicts a typical absorbance us. wavelength spectra for a 20- to 100-ug. range of concentration Recommended Procedure. Animal tissue has been chosen as the organic matter in the following description since it offers more of a challenge to the extraction technique. Common cosmetic or pharmaceutical creams and lotions are easier to handle. For determinations in animal tissue, the sample is cut into slices approximately ''*-inch thick. The slices are spread on Petri disies and placed in a vacuum oven with 26 inches or better raruum a t tempi2ratures of 30' to 35 O C. Up to 100 gra ns of sample can be used. Spray residues on peelings of fruits and vegetables would be treated in the same way. A small amount of air should be bled through the oven during drying. Dehydration usually takes about 38 hours. The dried residue is transferred to a high speed Blendor and pu1veriz:d for about a minute. After pulverizing, 50 ml. of 85% ethanol is added to the Blendor and mixed for about 5 minutes a t slow .peed to aroid solvent loss. The ethanol is decanted through filter paper into a glass joint Erlenmeyer flask. A second 50nil. extraction is made, and the extracts are combined. When the hexachlorophene content in liquids like blood or milk is investigated, there is no need for the blending step since the solids content would be low by comparison. In this case, after dehydration under vacuum. the solids are scraped loose from the Petri dish and slurried Kith 85% ethanol, warmed, and filtered in the same way into a flask. The ethanol is evapoApparatus.
415 WAVELENGTH,
400
700 rnp
Figure 1 , Typical absorption spectra of hexachlorophene concentrations with 4-aminoantipyrine reagent
rated using a gentle stream of filtered air Fifty ml. atapproximately 50" C. chloroform is added to the residue and warmed to dissolve the solids. Fifty ml. 98% sulfuric acid is then added to the flask containing the chloroform solution and the mixture is gently refluxed from 4 to 8 hours or until the chloroform layer is clear or light brown in color. After cooling, the layers are separated and the acid layer is discarded. The chloroform layer is washed neutral t o litmus, and drawn off into another separatory funnel. Three or more washings are usually necessary. The washed chloroform is evaporated. a h e n the last traces of solvent are gone, 20 ml. of ammonia buffer lis added, and the evaporating dish is kept warm to melt and dissolve any solidified residue. After adjusting the pH to 10.2 f 0.2 with 5% sodium carbonate, the spectrophotometric procedure described in the calibration curve preparation section is followed. RESULTS A N D DISCUSSION
I n developing this procedure, animal tissue and vegetables were injected with known amounts of hexachlorophene. Organs such as brains, liver, kidneys,
Table I.
and muscle of beef and lamb were obtained in local stores along with tomatoes, beans, and peppers representing common vegetables. Using alcoholic solutions containing 100 pg. of hexachlorophene per ml., known quantities were injected into the tissue specimens or dripped onto the surface of vegetables using a microhypodermic syringe. Attempts to extract hexachlorophene metabolized within tissue shows that it can be recovered. Only methods employing radioactive tracers could reveal the accuracy of recovery by the proposed method. These techniques have not been investigated. The scope and precision of our separation method are based simply on the sulfuric acid destruction of interfering materials as outlined. Table I illustrates the proposed method when used on the specimens listed. Values before and after treatment are included. The absorbance at 475 mp is also repoited together with the corresponding amount of hexachlorophene recovered. All specimen weights !yere conveniently chosen a t 25 grams. hIany techniques to recover and analyze the hexachlorophene were tried. Direct extraction of the tissue with hydrocarbon, oxygenated, and chlorinated solvents invariably ended up with emulsions difficult to break and with poor recovery of hexachlorophene. Since the tissue contains as much as 75% water, dehydration prior to analysis seemed advisable. Hexachlorophene could not be recovered if the tissue was dehydrated a t 100' C. or higher. The reason for this was not investigated. Two dehydrating techniques were found satisfactory: freeze drying and vacuum oven drying at 30" to 35' C. Most solvents extracted organic matter as well as hexachlorophene from the dehydrated tissue. Eighty-five per cent ethanol proved satisfactory, extracting only a minimum of organic
Examples of Recovery of Hexachlorophene from Various Specimens
Absorbance Specimen Green peppers Green peppers Cucumbers (peel only) Cucumbers (peel only) Tomatoes (peel only) Tomatoes (peel only) Peaches (peel only) Peaches (peel only) Blood serum (horse) Blood serum (horse) Whole blood (horse) Whole blood (horse) Brain (hog) Brain (hog) Liver (sheep) Liver (sheep) Kidney (beef) Kidney (beef) Whole milk Whole milk
at 475 mp 0 000 0 125 0 000 0 140 0 000 0 140 0 000 0 130 0 000 0 135 0 010 0 140 0 000 0 125 0 020 0 140 0 020 0 140 0.000 0 125
Hexachlorophene, ~ g . Added Found 0 25 0 25 0 25 0 25 0 2.5 0 25 0 25 0 25 0 25 0 25
VOL. 36, NO. 1, JANUARY 1964
0 24 0 29 0 20 0 26
o
27
2 20
0 24 4 29 4 29 0
24
125
material. The presence of extracted organic material would interfere with any subsequent determination. It is thus necessary to destroy the organic extractive with sulfuric acid, which is feasible, as hexachlorophene is stable under this treatment. The reaction seems t o be principally one of oxidation since charring is noted. The spectrophotometric method is sensitive to 20 pg. of hexachlorophene in animal tissue, as an example. In this range, the precision of the method is *lo% Of the present* The absorption for the ammonia buffer solution of the residue is a
straight line when no hexachlorophene is present, but it usually does not follow the zero absorption base line. A curve from 450-750 mp may be run on the ammonia buffer solution of the extract before the addition of 4-aminoantipyrine and potassium ferricyanide reagents t o compensate for any excessive baseline shift. LITERATURE CITED
M., J . Am. (1) Child% R. F.,Parks, Pharm. Assoc. Sci. Ed. 45,313 (1956). (2) Clements, J. E.,r\jewberger, S. H., J. Assoc. O$Lc. Agr. Chemists 37, 190
(1954).
(3) Gottlieb, S., Marsh, P. B., IXD. ENQ.
J.
c , p ~ ~ ~ ~ s ~ A ~ . E ~ : , 1 ~ ~ Pharm. &: Pharmacol. 1 0 , Supp. 171T
(1958).
( 5 ) Jmgermann, E., Beck, E.c.,J . AmOil Chemists SCC.38,513 (1961). (6) Hinge, K., Sei~en-Oele-FetteWache 85, 61 and 87, Feb. 4 and 18 (1959). (7) Larson, H. L., J . Assoc. Offic. Agr. Chemists 28, 301 (1951). (8) Lord, J. W., McAdams, J. A,, Jones, E. B., Soap Perfumery and Cosmetics
26,783 (1953):
(9) Pharmacopeia of the United States, Sixteenth Revision, 319-22, 1960.
(10) Sindar Corp., Technical Bulletin H-7.
RECEIVEDfor review April 16, 1963. Accepted October 18, 1963.
The Determination of Primary Alcohol Groups in Polyglycols Using Triphenylchloromethane JOE G. HENDRiCKSON
The Dow Chemicul Co., Freeport, Texas
F A new method developed for the analysis of primary alcohols in polyglycols i s based on the reaction of the polyglycol with triphenylchloromethane. The reaction was followed by the disappearance of the OH band in the infrared spectrum of 3280 cm.-' The reaction rate of triphenylchloromethane with primary alcohols i s 25 to 100 times faster than the reaction rate with secondary alcohols.
T
HE
REACTIVITY
OF
POLYGLYCOLB
used to produce urethane foams has been known to vary considerably from one lot to another. One important variable affecting the reaction rate is the amount of primary alcohol groups present. Recently, new analytical methods have been developed to measure small amounts of primary alcohol end groups in the presence of relatively large concentrations of secondary alcohol groups (where the total hydroxyl groups are about 2% of the total sample). Two of these are based on the faster reaction of the primary alcohols with either pyromellitic anhydride ( I O ) or toluene diisocyanate (3). However, both methods require the plotting of 10 to 20 determinations for the determination of the true rate curves. A third method recently reported, gives results in good agreement with the method of this report (6). Prior to the work of Siggia and Crummett, the chemical literature revealed no general method for the determination of nonvolatile primary and secondary alcohol end groups in poly126
ANALYTICAL CHEMISTRY
glycols. Oxidation followed by carbonyl compound analysis has been the usual approach (1, 6), but the further oxidation of ketones or esters, acetal hydrolysis, and ether cleavage would be expected under normal reaction conditions. The selectivity of primary and secondary alcohols reaction with triphenylchloromethane (trityl chloride) has often been reported (4-6, 8). This reaction has been used for cellulose analysis (9) in a method that required the isolation of the triphenylmethyl ether of the cellulose. However, following this reaction is quite difficult. Rate studies with trityl chloride have been based either on using radioactive alcohols (11) or on studying a reversible reaction ( 7 ) , and the reaction was frequently run for several days a t room temperature or for 24 hours with pyridine a t 70" C., for example, (4, 6). However, the variation of rate by using mixed solvents is common ( 7 ) . An infrared method is reported for the analysis of primary alcohols, using trityl chloride as the reactant. The reaction is followed by observation of the rate a t which the hydroxyl band disappears (at 3280 cm.-l). A rate plot is made and extrapolated back to zero time. The rate of disappearance of the secondary hydroxyl band is measured, and the quantity of primary alcohol is determined by difference. EXPERIMENTAL
Apparatus. A Perkin-Elmer 221-G double-beam infrared spectrophotometer was used with these settings:
interchange, grating; slit width, 0.927 x 2; suppression, 4; expansion, 1; attenuation, 11.0; scan program, i n ; scan time, 32: gain, 4 ; source, 0 . 3 ; gear ratio,'l.o. The flasks, hypodermic syringes, and needles were cleaned and dried a t 110' C. prior to use. Procedure. Runs were made in duplicate. Three samples were run in a 4- to 6-hour period. The described method keeps water vapor away from the samples and reactants. The reagent was prepared by diluting 5.0 grams of special grade trityl chloride (Fisher Scientific Co.) and 2.5 grams of tris (dimethyl amino) phosphine oxide (Darcol, Dow Chemical Co.) to 25 ml. with reagent grade quinoline (Fisher Scientific Co.). This mixture was shaken well and allowed t o stand for 1 hour. About 0.50 gram of sample was weighed into a 2-ml. volumetric flask and prepared reagent added to the mark. The flask was shaken vigorously by hand for more than a minute. A blank analysis was performed by making a scan with the reagent in both sample and reference cells. The sample cell was washed out first with reagent grade pyridine and then with chloroform. Next, the freshly mixed reactants were added to the sample cell. A scan between 3700 and 3100 cm.-I was made with the reagent in a cell in the reference beam, to determine the total absorbance of the hydroxyl band at 3280 cm.-1 The 2-ml. flask waa next stoppered with two syringe caps and placed in a constant temperature silicone oil bath a t 75" C. After an appropriate time, 0.2-ml. aliquots were removed through the serum cap with a hypodermic syringe. The samples were injected into the r
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