extract that was washed with methylene chloride under basic conditions to remove all the methylene chloride-extractable opium alkaloids from the aqueous phase, leaving morphine in the aqueous phase. Acidification of the aqueous sample solution before injection is necessary because the mobile phase is acidic (pH 4.8); injecting a basic sample solution into an acidic mobile phase may create chromatographic anomalies, such as band broadening, due to sample precipitation a t the column head. Figure 5 depicts a typical chromatogram of methylene chloride extract after the organic solvent was evaporated and the residue redissolved in the mobile phase. The reproducibility of the method is summarized in Table IV. The results were obtained by analyzing the same sample, prepared daily, for seven consecutive days. As can be seen, the two methods are quite precise for the determination of morphine and codeine. Somewhat lower precision was obtained for thebaine, since a relatively crude integrator was used for this work. Better precision for thebaine might be possible with. a more sophisticated data processor which could compensate, to some extent, for the poorer definition of the thebaine peak. Linear peak area response was obtained both at 254 and 286 nm for injection of up to at least 80 Fg of morphine, 25 pg of codeine, and 20 pg of thebaine. For the same quantities injected, only codeine gave linear peak height response. Precaution should be taken in this regard if peak height is to be used for quantitation. Although both 254 nm and 286 nm were used in this work, it is recommended that 286-nm detection be used whenever possible. The UV absorption spectra indicate that 286 nm is a common absorption maximum for the three major alkaloids in the mobile phase employed (Figure 6).
LITERATURE CITED (1)E. Stahl, Ed., "Thin-Layer Chromatography", Springer-Verlag, New York, N.Y., 1969,p 436. (2)D. Furmanec, J. Chromafogr., 89, 76 (1974). (3)A. Bechtel, Chromafographia, 5, 404 (1972). (4)K. D. Parker, C. F. Fontan, and P. L. Kirk, Anal. Chem., 35, 356 (1963). (5)S. Yamaguchi, I. Seki, S. Okuda, and K. Tsuda, Chem. Pharm. Bull., 10, 755 (1962). (6)H.A. Lloyd, H. M. Fales, P. F. Highet, W. J. A. VandenHeuval, and W. C. Wildman, J. Am. Chem. SOC.,82,3791 (1960). (7)C. Y. Wu, S.Slggia, T. Robinson, and R. D. Waskiewicz, Anal. Chim. Acta, 63,393 (1973). (8)J. H. Knox and J. Jurand, J. Chromatogr., 82,398 (1973). (9)J. H. Knox and J. Jurand, J. Chromatogr., 87,95 (1973). (10)P. J. Twitchett, J. Chromatogr., 104,205 (1975). (11) R . Verpoorte and A. B. Svendsen, J. Chromatogr., 100,227 (1974). (12)R. Verpoorte and A. B. Svendsen, J. Chromatogr., 100,231 (1974). (13)W. B. Caldweil, United Nations Secretariat, STISOAISER. J/17,(Nov.
. -. -,.
107A\
D. W. Smith, T. H.Beasley, Sr., R. L. Charles, and H. W. Ziegler, J. Pharm. Sci., 62, 1691 (1973). T. H. BeasleY, D. W. Smith, H. W. Ziegler, and R. L. Charles, J. Assoc. Off. Anal. Chem., 57,85 (1974). J. D. Wittwer, J. Forensic Sci., 18, 138 (1973). H. W. Ziegler, T. H. Beasley, Sr.. and D. W. Smith, J. Assoc. Off. Anal. Chem.. 57. 85 11975). (18)E. Murgia and H. F. Walton, J. Chromatogr., 104,417 (1975). (19)L. R. Snyder and J. J. Kirkland, "Introduction to Modern Liquid Chromatography", John Wiley and Sons, New York, N.Y., 1974,pp 239-281. (20)E. Brochmann-Hanssen, Medd. Norsk. Farm. Selsk., 17, 76 (1955). (21)E. Brochmann-Hanssen and A. B. Svendsen, J. Pharm. Sci., 52, 1134
(1963). (22)C. H.VanEtten, F. R. Earle, T. A. McGuire, and F. R. Senti, Anal. Chem., 28, 867 (1956). (23)United States Pharmacopeia, 19th Rev., Mack PublishingCo., Easton, Pa., 1975,p 350. (24)H. Bohme and R. Strohecker, Arch. Pharm., 285,422 (1952). (25)H. Bohme and R . Strohecker, Arzneim.-Forsch., 3, 236 (1953).
RECEIVEDfor review July 28,1976. Accepted November 24, 1976.
Quantitative Determination of StabiIizers in Tetraethylene Glycol Dimethacrylate by High Pressure Liquid Chromatography G. A. Pasteur Bell Laboratories, 600 Mountain Avenue, Murray Hill, N.J. 07974
A quantitative determlnatlon of 4,4'-thiobis(3-methyl-6-terfbutyiphenol) (TMTPB), methyl ether of hydroqulnone, and hydroquinone in tetraethylene glycol dimethacrylate (TEGDMA) has been achieved uslng liquid chromatography on 10-p porous silica particles and UV detectlon. interference due to polymerizationof TEGDMA induced by UV radiation from the detector is avoided by deflecting the UV beam when the preceding refractive index detector signals the elution of the monomer from the column. The detection limits are 15,9, and 21 ng for TMTBP, MEHQ, and HQ, respectlvely. Repllcate analyses of commercially available formulations had a precision (average deviation) of 5 % for concentrations above 50 ppm and 10% for concentrationsbelow 25 ppm.
Tetraethylene glycol dimethacrylate (TEGDMA) is used as a cross-linking agent in some polymer formulations ( I , 2). Hydroquinone (HQ) and methyl ether of hydroquinone (MEHQ) are used as TEGDMA polymerization inhibitors ( 3 ) which must be present a t high enough levels to ensure good shelf life and thermal stability during polymer processing, but
low enough not to interfere significantly with the cross-linking 4,4'-Thiobis(3-methyl-6-tert-butylphenol) reaction. (TMTBP) (Santonox) is the usual antioxidant included in TEGDMA. Commercially available TEGDMA must be characterized analytically to ensure that these protective additives are present at effective levels. Gas chromatography is not suitable for the analysis. Experiments a t the temperature necessary for elution and separation showed that TMTBP degrades and its degradation products obscure the chromatogram. Liquid chromatography (an ambient temperature process) has been chosen for this determination. A reliable analytical procedure for the quantitative determination of HQ, MEHQ, and TMTBP is described in this report.
EXPERIMENTAL T h e solvent delivery system is a Waters Associates M o d e l 6000 pump. A 25-cm Si-10 M i c r o p a k column (Varian) (1.5-mm id., 3.0-mm 0.d.) is used for t h e separations. T w o types o f syringes are used: 5-pl Pressure-Lok B-110 a n d 5 - p l H a m i l t o n 700 in conjunction w i t h a septum injector. A Waters Associates R 401 differential refractometer is used in series w i t h a V a r i a n 635 U V - v i s i b l e spectrophotometer
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Table I. Quantitative Determination of Stabilizers ~292,,~,
Stabilizer TMTBP RI
A SAMPLE
a
uv TMTBP MEHQ
STANDARD
Ha I
0
2
4
6
8 IO 12 14 ELUTION TIME (MIN.)
16
18
Flgure 1. Separation of 4,4'-thiobis(3-methy1-6-tert-butylphenol), methyl ether of hydroquinone, and hydroquinone in TEGDMA by liquid chromatography Column 25 cm X 1 5 mm Si-IO Micropak: eluant isooctane-ethyl acetatemethylene chloride, 7 6:1.2:1.2; flow rate 1 ml/min. Detectors: UV photometer, at 292 nm, for curve l a and 1b and RI for curve IC
equipped with an Aerograph Micro Volume flow cell assembly. Chromatograms were recorded on a dual pen Fisher Recordall 5000 recorder. All solvents are MC&B Spectrograde. Standards were analyzed quantitatively by mass spectrometry to determine their purity (greater than 98%).
METHOD OF ANALYSIS Standard solutions are prepared by dissolving appropriately weighed amounts of each stabilizer in 1:l ethyl acetate-isooctane and adjusting to a known volume. One-cll aliquots of these solutions are injected with the Pressure-Lok B-110 syringe for the best reproducibility. Separation of components is achieved by elution with isooctane-ethyl acetate-methylene chloride, 7.6:l.Z:l.Za t a flow rate of 1 ml/min. The variable wavelength UV detector is set at 292 nm which is the wavelength of maximum absorption for the hydroquinones. Calibration curves are then established by plotting peak heights in absorbance units vs. concentrations in ppm units for each stabilizer. One-pl aliquots of commercial TEGDMA containing unknown amounts of stabilizers are injected with the Hamilton syringe. To prevent plugging of the syringe by any polymer formed, it should be thoroughly cleaned immediately after each injection with a 50% mixture of ethylacetate and tetrahydrofuran. Because of the high viscosity of TEGDMA, good reproducibility is achieved without the use of high pressure syringes. For reasons which are described later, the UV beam is deflected away from the UV cell when the refractive index (RI) detector, placed ahead of the UV detector, signals the passage of TEGDMA. RESULTS AND DISCUSSION Figure l a shows the separation obtained for a standard solution containing 960 ppm of TMTBP, 50 ppm of MEHQ, and 120 ppm of HQ. Calibration curves were established from peak height measurements on chromatograms obtained from 3-pl injections of standard solutions of the stabilizers. The concentration ranges of stabilizers in the standards were chosen to be similar to the level nominally present in com-
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1. mole-1 cm-l 5800
MEHQ
3300
HQ
3300
Detection Concentration limit, Sample f av dev, ng No. PPm 15 1 980 f 40 2 1050 f 40 9 1 27 f 2 2 10 f 1 21 1 55 f 2 2 172 f 5
mercial TEGDMA, namely 950 to 1700 ppm for TMTBP, 120 to 240 ppm for HQ, and 50 to 140 pprn for MEHQ. Table I shows the results obtained for the analysis of two commercial samples of TEGDMA containing the three stabilizers. The detection limit was defined as the amount of stabilizer producing the smallest detectable signal from the UV detector which was 0.001 absorbance unit. Using 3-111 injections, concentrations as low as 5 ppm for TMTBP and 3 ppm for MEHQ and 7 ppm for HQ could be determined. The detection limits are 15 ng, 9 ng, and 21 ng for TMTBP, MEHQ, and HQ, respectively. The higher detection limit for HQ is due to a band broadening effect. The arithmetic average of deviations is computed on a minimum of four injections for each sample. For informative purposes the molar absorptivities, 6, at 292 nm are also listed. Figure l b and ICare simultaneous UV and RI chromatograms of stabilizers in a commercial TEGDMA sample. The amounts of TMTBP, MEHQ, and HQ in this sample are found to be 1050 f 40,lO f 1, and 170 f 5 ppm, respectively. Polymerization of TEGDMA is induced by UV irradiation (4) and visible light ( 3 ) .Even though the molar absorptivity of TEGDMA is low a t 292 nm, it is not so low as to preclude the possibility of polymerization in the UV detector cell. For example, repeated injections of the monomer result in enough polymerized material in the cell which leads to a substantial reduction in the effective cell volume or transmitted light intensity. This precludes quantitative analysis. Therefore, in order to avoid polymerization the UV beam was deflected away from the UV cell during the passage of the monomer. It was possible to use this technique since measurements of the TEGDMA peak height in the UV chromatogram were not needed for calibration. An RI detector is placed ahead of the UV detector and the deflection of the UV beam is synchronized with the RI signal obtained from TEGDMA. On Figure I b the square wave signal at 12.3 min represents the deflection of the UV beam. A small correction (0.35 min) is made for the time of passage between the two detectors.
ACKNOWLEDGMENT I thank D. J. Freed for fruitful discussions. LITERATURE CITED (1) W. A. Salmon, and L. D. Loan, J Appl. Polym. Sci., 16, 671 (1972). (2) J A. Manson and L. H Sperling, "Polymer Blends and Composites", Plenum Press, New York, 1976, p. 226. (3) E C Leonard, "Vinyl and Diene Monomers", Part I, Wiley-Interscience, New York, 1970, p. 168 (4) M. Dole, "The Radiation Chemistry of Macro-Molecules", Academic Press, New York and London, 1973.
RECEIVEDfor review September 10,1976. Accepted December 2,1976.
