standard deviation of the Monte Carlo X,may be a crude It approaches measure of the uncertainty associated with X,. the well-understood meaning as the distribution of X , approaches normality. A reviewer suggests t h a t a simple test should be devised to help an analyst decide if the deviation from normality in X , is sufficient t o warrant using the more sophisticated Monte Carlo method over the local linearization method. One possibility is t o draw the calibration curve and as in Figure 1to lay off ordinates Y L and 2 YlZat, say, 2 s y d n tervals each from the mean yL.Reading from the curve X(Y,), X ( Y L 1and ) X(Y , J , the analyst has a measure of skewness of the X distribution as the difference between X ( y Land ) 112 [ X ( Y , , ) X ( Y , , ) ] as is shown in Figure 1. This simple test ignores the effect of the scatter of y, on the X distribution and hence should be used with caution when this scatter is large.
LITERATURE CITED C. A. Bennett and N. L. Franklin, "Statistical Analysis in Chemistry and the Chemical Industry", John Wiley and Sons, New York, 1954, sec. 6.24. i. M. Koithoff, E. B. Sandeil, E. J. Meehan. and S. Bruckenstein, "Quantitative Chemical Analysis", Fourth ed., Macmillan, New York, 1969, Chapter 16. G. E. Forsythe, J. SOC.lnd. Appl. Math., 5 (2),74 (1957). G. Henderson and A. Gajjar. J. Chern. Educ., 48,693 (1971). 0. L. Davis and P. L. Goldsmith, "Statistical Methods in Research and Production", Fourth revised ed., Oliver and Boyd, Edinburgh, 1972, Chapter 0. A. Raiston, "A First Course in Numerical Analysis", McGraw-Hill Book Co., New York, 1965. L. M. Schwartz, Anal. Chern., 47, 963 (1975).
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RECEIVEDfor review May 13,1976. Accepted September 7 , 1976.
Vapor Phase Silylation of Laboratory Glassware David C. Fenitnore,* Chester M. Davis, James H. Whitford, and Charles A. Harrington Texas Research lnstitute of Mental Sciences, Texas Medical Center, Houston, Texas 77030
Irreversible adsorption of sample material on the surfaces of laboratory glassware is a problem encountered frequently in determinations of microgram and submicrogram amounts of polar compounds. The severity of the problem can be such as to constitute the major contributing factor limiting the sensitivity of certain assay procedures. For this reason many laboratories, particularly those involved in assays of samples of biological origin, inactivate the surfaces of glassware used in sample preparation by treatment with various silylating reagents. These procedures, which are often used for inactivation of gas chromatographic column materials ( I , 2), usually involve treatment of the glass surface with hexamethyldisilazane, dimethyldichlorosilane, or mixtures of these or similar silylating reagents dissolved in solvents such as toluene or pyridine. The glassware is then rinsed in additional solvent followed by further rinsing with methanol to hydrolyze any unreacted dimethyldichlorosilane if that compound is used in the reagent mixture. Such treatment produces a very satisfactory nonadsorptive surface, but where large amounts of glassware are processed, the excessive volumes of solvent employed create problems of disposal or reclamation. We therefore developed a glassware silylation procedure similar to that used by some investigators in preparing glass capillary columns for gas chromatography ( 3 ) . T h e treatment utilizes small amounts of hexamethyldisilazane in the vapor phase a t elevated temperature and completely eliminates the need for solvents in the process.
I
Chloromethylsilanes are not required as catalysts under these conditions which also eliminates problems arising from formation of hydrogen chloride. Large amounts of glassware can be prepared with minimal manipulation, low cost, and absence of environmental hazards.
EXPERIMENTAL Vapor phase silylation was performed in a vacuum oven (Blue M Model POM-16VC-2) evacuated by a mechanical vacuum pump (Welch Duo-Seal Model 1402) with the exhaust vented to the laboratory fume hood system. The inlet port to the oven was modified with standard pipe and tube fittings to permit the attachment of a small glass reservoir with an internal fritted glass gas dispersion element for vaporization of the silylating reagent (Figure 1). Atmosphere admitted to the inlet of the tube passed through a drying tube containing a desiccant. Reagent. Hexamethyldisilazane (HMDS) was obtained from Pierce Chemical Company, Rockford, Ill., and was used as received. Procedure. Glassware was washed thoroughly using a commercial Apparatus.
HMDS
Figure 1. Tube with fritted glass element for introduction of hexamethyldisilazane vapor to vacuum oven
Figure 2. Recovery of AQ-tetrahydrocannabinol-3Hfrom silylated ( 0 ) and untreated (0)glassware. Vertical lines indicate standard deviation of six determinations measured as disintegrations per minute (DPM)
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
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PENTOBARBITAL ( + q I
Flgure 3. Gas chromatographic determinations of pentobarbital in human blood samples using glassware for sample preparation which solvent silylated (0),vapor phase silylated ( O ) , and untreated
was
(A)
laboratory detergent (Alconox), rinsed in dilute hydrochloric acid, tap water, and deionized water. The clean glassware was then placed in the vacuum oven in commercial aluminum glassware baskets, and with the oven inlet open the oven temperature was raised to 200 "C. With the inlet valve closed, the oven was then evacuated for approximately 10 min. The evacuation valve was closed and the HMDS reservoir was connected to the oven inlet. The inlet valve was carefully opened just to the point where atmosphere was slowly admitted carrying HMDS vapor into the oven. After a pressure rise in the oven of approximately 5 in. of water, i.e. the vacuum was at 25 in., the inlet valve was closed and the glassware remained in the partial HMDS atmosphere for approximately 1h. The oven was then re-exhausted to remove unreacted HMDS and ammonia, the sole reaction product, and air then was readmitted slowly with the oven heater and vacuum pump turned off. After cooling, the silylated glass may be used immediately or stored for future use.
RESULTS AND DISCUSSION The performance of glassware silylated by vapor phase and solvent procedures was compared to untreated glassware in various assays where problems of surface adsorption had been experienced previously. The binding of Ag-tetrahydrocannabinol (A9-THC) t o glass surfaces which had undergone pre-treatment with silylating reagents and water-soluble silicone was examined by Garrett and Hunt ( 4 ) who found considerable adsorption of this compound t o both untreated and water-soluble silicone-treated glassware. Silylation of the glassware, however, reduced the binding of the compound significantly. In the present study A9-THC3H in amounts ranging from 0.25 to 50 ng were dissolved in 1 ml hexane in borosilicate glass culture tubes and mixed with 1 ml water using a vortex mixer. After centrifugation, 0.5-ml aliquots of the hexane layer were transferred to counting vials for measurement of the recovered THC. Figure 2 shows the comparison of vapor phase silylated and untreated glassware. Not only is recovery incomplete but also the variability of recovery is quite high in the untreated glassware. In determinations of pentobarbital in blood serum by gas chromatography, which requires solvent extraction and concentration of the extract prior to gas chromatography, the sample is exposed t o glass surfaces not only in the extraction tubes but also in transfer pipets and concentration vials. Figure 3 shows flame ionization detector response to various amounts of pentobarbital added to human blood serum and processed in treated and untreated glassware. Glassware silylated by both solvent and vapor phase procedures produced identical linear relationships between peak height and
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LITERATURE CITED (1) E. C. Horning, E. Moscatelli and C. C. Sweeley. Chem. I d . , 751 (1959). (2) J. Bohemen, S. H. Langer, R. H. Perrett, and J. H. Purnell, J. Chem. Soc., 2444 (1960). (3) M. Novotny, L. Blomberg, and K. D. Bartle, J. Chromsfogr. Sci., 8, 390 (1970). (4) E. R. Garrett and G. A. Hunt, J. Pharm. Sci., 63, 1056 (1974). (5) M. Kutas, A. Chung, D.Bartas, and A. Castro, Steroids, 20, 679 (1972).
RECEIVEDfor review June 21,1976. Accepted September 9, 1976. This investigation was supported in part by National Institute of Drug Abuse Research Grant No. 5 R01 DA 00093-06.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
