(3) Dubois, L., Corkery, A,, Monkman, J. L., Inter. J. Air Pollution 2, 236 (1960). (4) Hoffman, D., Wynder, E. L., Cancer 15,93 (1962). (5) Kotin, P., Falk, H. L., Mader, P.,
Thomas, M., A . M . A . Arch. Ind. Hygiene 9, 153 (1954). (6) Kotin, P., Falk, H. L., Thomas, M., Ibid., 9,164 (1954). (7) Lyons, M. L., Johnson, H., Brit. J. Cancer 11, 60 (1957). (8) Sawicki, E., Hauser, T. R., Elbert,
W. C.. Fox. F. T.. Meeker. J. E.. Am. Ind. Hyg. Assoc. Quart. 23,'137 (1962). (9) Spotswood, T. M., J. Chromatog. 2, 90 (1959). (10) ibid., 3 , 101 (1960). (11) Tebbens, B. D., Thomas, J. F.,
Mukai, M., A.M.A. Arch. Ind. Health
13,567 (1956). (12) Ibid., 15, 413 (1956). (13) Tebbens, B. D., Thomas, J. F., Sanborn, E. N., Mukai, M., Ibid., 17, 152 (1958).
(14) Thomas, J. F., Tebbens, B. D.,
Mukai, M., Sanborn, E. N., ANAL. CHEW29,1835 (1957). RECEIVED for review Kovember 4, 1963. Accepted February 17, 1964. Work supported by the Division of Air Pollution, Bureau of State Services, Public Health Service, and is a cooperative effort within the University of California of the School of Public Health and the Sanitary Engineering Research Laboratory, Department of Engineering. Division of Water and Waste Chemistry, 144th Meeting, .4CS, Los Angeles, Calif., April 1963.
Identification of Trace Quantities of Antioxidants in Polyethylene D. F.
SLONAKER and D. C. SEVERS
Research laboratories, Tennessee Eastman
b The identification of trace quantities of phenolic antioxidants in polyethylene has been hampered in the past by difficulties in separating the antioxidant from other soluble components in the polymer. A method has now been developed for isolating and identifying quantities as low as 1 p.p.m. of a phenolic antioxidant in polyethylene. This method can be used when several antioxidants, including synergistic mixtures, are present and/or when proof of the absence of toxic or harmful antioxidants is necessary. The method involves extracting the antioxidant with hexane a t 50" C. and filtering the extract at 0" C. The extract filtrate is concentrated by evaporation. The solution is again filtered and concentrated before being extracted with ethyl alcohol. The alcohol solution is treated with phosphomolybdic acid and ammonia. The formation of a blue color confirms the presence of a phenolic antioxidant. Thin layer chromatography of the alcohol solution can be used to separate mixtures of antioxidants and qualitatively identify them. Up to four different antioxidants have been extracted from a polyethylene and identified in this manner.
A
is one of the most heat stable plastics, it is advantageous to add trace quantities of antioxidants to formulations which are to be extruded and molded at relatively high temperatures. Phenolic antioxidants may be used in polyethylene for this purpose in concentrations as low as a few parts per million. At present, these antioxidants cannot be identified directly in polymer compositions containing several such compounds if their concentration is very LTHOUGH POLYETHYLENE
1130
ANALYTICAL CHEMISTRY
Co., Division of Eastman Kodak Co.,
Kingsport, Tenn.
low, but must be removed by extraction. Such extracts also contain low-molecular-weight wax, which must be removed because it changes the R, value of the antioxidant on the chromatogram. This work is concerned with the development of an improved method for isolating the antioxidant from the polymer and for determining its composition. Paper, as well as thin layer chromatography, is used to separate the phenolic antioxidants, which are in solution. Phenols that do not react with phosphomolybdic acid can be located on thin layer chromatograms by spraying the chromatogram with concentrated sulfuric acid containing 5% nitric acid. The chromatographic separation of phenolic antioxidants has been studied by several workers (1-3, 5-79, Schmidt and Bonomo, of Wright Air Development Center, issued a very comprehensive technical report concerning the isolation and identification of additives in synthetic lubricants (4). However, such previously outlined methods do not enable the separation of the antioxidant from the low-molecular-weight wax, and therefore cannot be used t.0 identify trace amounts of antioxidants in polyethylene. The method herein described provides the necessary isolation and identification of antioxidants by a single routine procedure that is applicable to all polyethylenes. EXPERIMENTAL
Isolation and Identification Procedure. The procedure for extracting and identifying antioxidants in polyethylene is simple and straightforward. The equipment is of a basic nature common to the field of chromatography. The antioxidants are first extract,ed from 1 kg. of polyethylene in the form of granules or chopped film with hexane
a t 50" C. in a 3-liter1 round-bottomed flask. Figure 1 shows this step as well as successive steps in the separation procedure. After the warm extract solution is decanted, filtration a t 0" C. is sufficient to remove a large part of the polyethylene wax. A 600-ml., sintered-glass funnel having a mediumporosity frit is used with the aid of vacuum for this filtration. Since some of the lower-molecular-weight wax is soluble in hexane a t 0" C., the filtrate is then concentrated to 35 ml. by allowing it to evaporate slowly (not exceeding 50' C.) on a steam bath. This concentrate is again cooled and filtered a t 0" C. The resulting filtrate is then slowly concentrated to about 5 ml. Care must be taken not to evaporate to dryness in order to minimize the loss of volatile antioxidants, such as BHT (2,6-di-t-butyl-p-cresol, Eastman Chemical Products, Inc.). The antioxidant can now be extracted from the wax-containing concentrate with two 5-ml. portions of ethyl alcohol. The alcohol is slurried with the hexane containing the wax and antioxidants a t about 50" C. Upon cooling, the alcohol layer can be decanted, while the hexane layer containing the very low molecular weight polyethylene remains adhered to the container in the form of a greasy semisolid. In some cases, filtration of the final antioxidant solution through relatively coarse filter paper is necessary to remove small droplets of residual hexane and wax. Known solutions of antioxidants are prepared by dissolving 100 mg. of each compound in 10 ml. of ethyl acetate. When the paper chromatographic separation is used, small spots of both the known ethyl acetate solutions and the unknown ethyl alcohol solution are applied a t intervals along a line near the bottom of the paper. Enough is added to give 20 to 50 wg. of the compound in each spot. The paper is then dipped into a benzene solution of cotton seed oil up to, but not touching, the spots. After the benzene has been allowed to
i5 L. AEXANE
[>OT.,,4
HR]
(DISCARD)
I
p c . FILTRATIO~
F
I
l
HEXANE SOLUBLE
Figure 2. Paper chromatographic separation FILTRATE
IDISCARD)
I
TWO 5-ML EXTRACTIONS [WITH ETHTL ALCOHOJ I
TRACE POLYETHYLENE WAX
IN ETH,YL ALCOHOL
IN CYCLqHEXANE
Figure 3. Thin layer chromatographic separation
Figure 1. Flow diagram of antioxidant isolation and identification
evaporate, the sample end of the paper is placed in a solvenl, trough in a chromatography jar containing an 80/20 v./v. solution of methanol and water. The chromatogram is allowed to develop in the ascending direction until the solvent front is about an inch from the top of the paper. The paper is then removed from the sealed jar and sprayed with a 3% solution of phosphomolybdic acid in ethyl alcohol. It is then !uspended in a jar containing ammonium hydroxide. Blue spots appear wherever antioxidant is locatad on the paper. The spots can be made more distinct by respraying the paper and re-exposing it to ammonia vapors. Since the spots fade slowly with time, it is desirable to photograph the chromatogram t o obtain a permanent record. Such a photograph is shown in Figure 2 in which the separation of AC-5 12,2'-methylenebis(6 t - butyl - p - cresol)], Catalin Corp., WSP {2,2' - methylenebis[6 - (1methylcyclohexyl) - p -cresol]], Amold, Hoffman & Co., Inc., and Santonox R [4,4'-thiobis (&&butyl-m-cresol) 1, Monsanto Chemical Co,, from a known mixture is shown on oil-coated paper. Thin layer plates are prepared by thoroughly mixing a sufficient amount of Silica Gel G with twice its weight of water. This must be done in less than 2 minutes as the mixt .re sets up rapidly. The slurry is poured onto the roughened side of a glass plate and spread evenly by using a straight edge. The plate is allowed to stand until hard and then
-
in the sample were isolated according t o the procedure used for unknown polyethylene extracts. Antioxidants AC-5, WSP, and Santonox R could be detected in concentrations as low as 1 p.p.m.; however, B H T could not be detected in concentrations lower than 6 p.p.m. This is probably due t o the rather high volatility of BHT. This still left unanswered the question of whether the antioxidants could be extracted and identified in polyethylene containing a mixture of these antioxidants in trace quantities. T o answer this, an experiment was conducted in which 1 p.p.m. each of AC-5, WSP, and Santonox R was milled into a partially extracted sample of a commercial polyethylene; 6 p.p.m. of BHT was also milled into the polyethylene. The polymer was then granulated and extracted, and the antioxidants were isolated. A thin layer chromatogram of the final alcohol solution gave spots having RI values corresponding to AC-5, Santonox R, WSP, and BHT, as shown in a photograph of the chromatogram in Figure 4. A control prepared from the same polyethylene not containing the four known antioxidants is also shown along with a separate chromatogram of each antioxidant in order to compare the R/ values, Several additional spots on the sample chromatogram are also seen on the chromatogram of the control, which indicates that the starting polymer still contained extremely small quantities of several unknown antioxidants. DISCUSSION AND CONCLUSIONS
Figure 4. Identification of antioxidants in extract
in an OveIl at 1150 C. for an hour. The sample is applied to the plate in the same manner in which it is applied to paper except that more Sample can be loaded onto the plate. The developing solvent mixture contains 47, v./v. methanol in cyclohexane. The chromatogram is developed in the ascending direction in a sealed chromatography jar. Phosphomolybdic acid is again used as the color-developing agent. Figure 3 is a photograph of a thin layer chromatogram in which AC-5, BHT, WSP, and Santonox R were separated from a known mixture of the four antioxidants in ethyl acetate. Application of Procedure. An experiment was carried out t o determine the minimum amount of each of several common antioxidants that could be isolated and detected. A known amount of each antioxidant was added t o a hexane solution of polyethylene wax. The antioxidants
The procedure described for isolating and identifying antioxidants present in trace amounts in polyethylene is superior to other methods in that it gives almost complete separation of the antioxidants from the polymer wax. Other methods of separating the antioxidant from the polyethylene, such as Soxhlet extraction, proved inefficient. Such extractions with polar solvents often do not swell the Dolvmer sufficiently to remove the antioxidant. Single-step extractions wit,h nonpolar solvents remove the antioxidants as well as some polyethylene wax. The antioxidants might be extracted more quantitatively from polyethylene a t higher than 50" C.; however, lowmolecular-weight polyethylene would also be extracted, which would in turn add to the isolation problem. Most of the polyethylene wax is insoluble in hexane a t 0' C., whereas the antioxidants retain their solubility a t this temperature. This allows separation of the antioxidant from the wax by means of filtration a t reduced temperatures. For analyzing the final antioxidant solution by paper chromatography, oilcoated papers are most effective. However, a larger sample loading can be used with thin layer chromatography than with paper chromatography. For this VOL. 36, NO. 6, M A Y 1964
1131
reason, thin layer chromatography was adopted as the standard procedure. A solution of phosphomolybdic acid followed by ammonia is used as the color developer. With this method, it is possible to isolate and identify phenolic antioxidants present in polyethylene in concentrations as low as 1 p.p.m. At present this technique has been applied primarily to the identification Of phenolic-type antioxidants in polyethylene;
however, preliminary indications are that it can be applied to the analysis of antioxidants in other polyolefins. LITERATURE CITED
(1) Dehority, B. A,, J. Chromatog. 2,384387 (1959). (2) Luongo, J. P., ANAL.CHEM.33, 1817 ( 1961 ).
(3) Mitchell, L. c., J. A S S O C . OflC.Agr. Chemists 40, 909 (1957). (4) Schmidt, J. J. E., Bonomo, F. S., “Development of Schematic Analytical
Procedures for Synthetic hbricants and
$ !:5‘4 4 6 4 , 114-150, ~
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RECEIVEDfor review June 3, 1963. Accepted February 19, 1964. Presented at Southeastern Regional Meeting, ACS, Gatlinburg, Tenn., Nov. 1-3, 1962.
Rapid Spectrophotometric Method for the Determination of Lead in Bone Ash FRANK H. ILCEWICZ, RICHARD B. HOLTZMAN, and HENRY F. LUCAS, Jr. Radiological Physics Division, Argonne National laboratory, Argonne, 111.
b The measurement of the absorbance of the chloro-complex of lead(ll) in 9M hydrochloric acid at 271 mp provides a simple and rapid method for the analysis of microgram quantities of lead in bone ash. Interfering ionse.g., iron(ll1) and copper(l1)-were removed by extraction with triiso-octylamine. The effect of the concentration of hydrochloric acid and calcium phosphate (bone ash) on the absorbance was studied. The optimum experimental condition was attained when bone ash was dissolved in 9M hydrochloric acid at a concentration of 0.2 gram ash/ml. of acid. Beer’s law was obeyed for lead concentrations up to 16 pg./ml. The sensitivity was 0.1 pg./ml. and the standard error for calcium phosphatelead standards was 3=270. The procedure has been used for determinaticns of lead in samples of bone ash in which the concentrations ranged from 6 to 80 p.p.m.
T
of a simple and accurate method for the determination of lead in bone specimens is extremely important in view of the high toxicity of the element and its wide distribution. It is also important for studying the metabolism of the element and comparing its distribution to that of lead-210, a decay product of radium226, in the human skeleton, which has already been studied in this laboratory (4,5). Lead is found in all tissues and fluids of the body and its distribution has been investigated extensively by many workers. For bone samples, spectrographic (IO), polarographic ( I ) , and colorimetric (7‘) methods have been employed for its determination. The HE DEVELOPMENT
1 132
ANALYTICAL CHEMISTRY
cupric, mercuric, and stannic chlorides, bismuth trioxide, cadmium, and thallous nitrates, and tellurium, and zinc metals in 9M HC1. The amount of nitrate introduced in the thallium and cadmium studies was assumed t o be insignificant. DIETHYLAMNONIUM SALT OF DIETHYLDITHIOCARBAMATE (DDC). One gram of DDC was dissolved in 100 ml. of chloroform. TRIISO-OCTYLAMINE (TIA). A 20% v./v. solution of TIA was prepared in chloroform and washed twice with equal volumes of 9M HCl. CHLOROFORM. All chloroform used to wash reference and sample solutions was equilibrated twice with equal volumes of 9M HC1. REFERENCE SOLUTION.One hundred milliliters of 9M HC1 was extracted three times with 5 ml. of TIA and washed three times with 5 ml. of chloroform. CALIBRATION SOLUTION.(0.20 gram of Pb-free synthetic bone salts per ml. of 9M HC1.) Lead-free synthetic bone solution was prepared by extraction of Reagent Grade calcium phosphate with 1% DDC in chloroform (7). It was then evaporated to dryness and ashed overnight a t 620’ C. in a Vycor beaker to remove any residual organic material. The ash was dissolved in 9M HC1. Any residual carbonaceous material was removed by filtering through a previously washed glass-fiber filter. The EXPERIMENTAL solution was brought to a voiume calculated to give a concentration of 0.20 gram of Caa(POa)aper ml. of 9-If HC1. Spectrophotometers. The measureThis solution (100 ml.) was then exments were made on a Perkin-Elmer, tracted three times with 5 ml. of 20% Model 350 recording spectrophotomTIA and three times with 5 ml. of eter and a Beckman DU spectrochloroform. The absorption spectrum photometer with 1.0-cm. silica cells. Reagents. STANDARD SOLUTIONS. of the solution showed no detectable amounts of lead (
