Estimation of Submicrogram Quantities of Chrysotile Asbestos by Electron Microscopy Anthony L. Rickards TBA Industrial Products Ltd., P. 0.Box 40, Rochdale. England
An extensive study has been undertaken at T B A Industrial Products Ltd., sponsored by the Asbestosis Research Council, into techniques for measuring t h e chrysotile asbestos content of the environment. Preliminary results were reported by Rickards a n d B a d a m i (1) a n d t h e X-ray diffraction methods employed i n t h e initial studies have been described by Rickards (2). However, none of t h e samples collected in t h e immediate neighborhood of a large asbestos textile factory contained sufficient chrysotile for analysis by t h e X-ray method-ie., t h e chrysotile asbestos content was less t h a n 0.1 pg/m3 of air. Hence, a more sensitive electron microscopic method for estimating chrysotile i n air has been developed. T h e method is based on the ability t o recognize small fibers of chrysotile i n t h e electron microscope by their unique morphology a n d electron diffraction patterns. Quantitative estimates are made by directly observing t h e dimensions of chrysotile fibers i n t h e samples and using t h e theoretical density of chrysotile to calculate t h e mass of chrysotile i n t h e sample. T h e technique is capable of measuring down to 0.1 ngJms (10-10 g/m3) and samples from several rural and urban sites have been examined, including a traffic intersection in a n industrial town center.
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Figure 1. Chrysotile 002 X-ray diffraction maxima from 100 pg of chrysotile on membrane filters confirming that chrysotile is not decomposed during ashing at 450 'C fa) before ashing. fbi alter ashing
EXPERIMENTAL Sample Pre-Treatment. Samples of airborne solids, collected with a Litton high volume sampler from typically loo0 mJ of air, were filtered onto B 47-mm cellulose nitrate membrane filter (mean pore diameter 0.05 pm). In order to improve the filtration rate, the samples of solids were first coagulated by adding sufficient sodium chloride to the samples to give a 10% solution. After filtration, the samples were dried and weighed; a typical sample weighed 100 mg. A parallel blank sample was prepared simultaneously, using membrane filtered deionized water and treated in a manner identical to the real samples. The solids were recovered from the membrane filter bv ashinc
1
of chrysotile were lost, probably due to the explosive combustion of the membrane. This danger was removed if the membranes were wetted with dibutyl phthalate before ignition, as can be seen from the X-ray diffraction traces before and after ignition (Figure 1). The membrane filter containing the sample was inserted into a 10-ml borosilicate glass tube. Ashing was carried out by inserting the tubes containing the samples into cavities in B capper block. thus eliminating any possibility of asbestos contamination from furnace insulation. The block was heated up to 450 "C by a heating plate and the temperature monitored with a thermoeou. . inro . . me cemer . 01 me DIOCK. I ne samples were poslpie insenea timed in the cold block and allowed to heat up over a period of 1 hour. At 200 'C, the membrane filter decomposes and at about 400 "C, the dibutyl phthalate starts to distil off. The samples were held at 450 'C for two hours, this being sufficient to burn off the organic material from most samples. Throughout the ashing procedure, the tubes were open to air. To avoid sample contamination with dust particles from the ambient air in the laboratory, the heating block was enclosed under a borosilicate glass hood (300-mm diameter) which was supplied with membrane filtered,
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(1971)
Figure 2. Electron micrograph of chrysotile after ultrasonic treatment illustrating the reduction of chrysotile fibrils to an approximate length 01 1 pm compressed air. The positive pressure generated around the heating block removed the possibility of serious eontamination of the samples and led to ultimate "blank" values of typically 12-11 fibrils per grid square as against about 50-11fibrils per grid ;quare in the absence of such a hood. The ashed samples consisted of a loose brown residue (typically 20 mg) a t the base of the tube, Immediately upon cooling, 5 ml of filtered deionized water were added to the samples and the tubes sealed with plastic caps. The tubes were then suspended in an ultrasonic water bath operating at 45 kHz, 100 W tuned to maximum resonance for a minimum period of 4 hours. This process caused agglomerated particles to be broken down and separated any chrysotile present into ultimate fibrils. It has been found that using the conditions described above, chrysotile fibers are generally broken up into fibrils approximately 1 pm long (Figure 2). In addition to breaking down large particles and fibrils, the ultrasonic treatment is a very effective method for homogenizing the suspension, an essential requirement for a reliable quantitative estimation. The samples were diluted to an appropriate concentration, depending on the total amount of solids, from which electron microscopic samples could be prepared. The sample already contained in 5 ml of water was suspended in 100 ml of filtered deionized water. The blank samples were also diluted by the same factor. After dilution, the samples were.~ given a further ultrasonic treat. .. ment tor U hours to ensure eomdete disinteeration of aeelomerated particles. ANALYTICAL CHEMISTRY, LvL.
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tile fibrils has shown that in spite of the differences in their geosize distri.graphical . . . . . .origin, . . the fibrils have . .a surprisingly . . . . . narrow . .. DUtlOn (3) with a mean standard deviation of 10 nm from elght different chrysotiles. On the basis of these two observations, the quantitative estimation of ehrvsotile entails the measurement of the total length of the fihrilsand subsequent conversion to volume and then to I"ass The fields of vi ew recorded onto photographic plates were eval"nP111n1 *+" I R Y m.o.rifi*.+in" uated using a hi . ... ..tereomirmrmn. ............... r_ _ The plates were systematically scanned and a value obtained for the total length of chrysotile fibrils, using a graticule eyepiece. The distribution of chrysotile fibrils on the grid square was recorded in the following manner. A graticule was constructed of 13 X 13 lines spaced at 3-mm intervals so that each square eorresponded to the field of view of the stereomicroscope. The graticule was laid on top of a plate which was in turn laid on the stage of the microscope and aligned so that the x / y traverse of the miemscope corresponded with the I and y directions of the graticule. Using this graticule, areas on the plate could he given I,y coordinates. During the measuring process the plate was systematically scanned, measuring the length of chrysotile fibrils in each graticule square. Ultimately a record of the fibril length in each graticule square was recorded. This was repeated for all the plates, including tbaae from the blank specimen. Finally, an average value for length of chrysotile fibrils per grid square was obtained. " , ~ .. . .O me .~ . manK . . .value . . nom . . .tnac . .oocanea . . . .from . tne real D U D T ~ B C ~01 samples gives the corrected average length of fibrils per grid square. Multiplying by the dilution factor aives the ehrvsotile content of theoriginal total sample in terms 07 length. Finaily the total length of chrysotile fibrils is converted to B mass value using the average fibril diameter of 340 A and the theoretical density of 2.55 ", eIem3. It has been found convenient to sample from lo00 m3 of air which when udnlr t.h.. o.1 ittnn hich vo+i>rno ramnlor *nn h m *.-id" .................... ._.I...I. -...r_"_ "_ out over a two-hour period. Some typical results obtained from various sampling sites are given in Table I. The statistical significance of the fibril counts, in relation to the reference blank specimens, has been assessed by a special statistical procedure which will be described in a separate publication (4). Significance tests have been applied to all the results quoted in Table I and show that they have significance vahes ~~~~~~~
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Figure 3. Electron micrograph of a field of view of a sample of airborne solids. Magnification X2000 The insert is a detail of two fibrils in the sample. Magnification X20.000 Specimen Preparation for Electron Microscopy. Samples for electron microscopy were prepared by drying a drop of the homogenized suspension onto a carbon-coated 4W-mesh electron microscope grid. The volume of the drop, which must he k n o m
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fnr o + m , , i n d_"dn0 L. .....t.h .e final n,,antitative ~ s l n n t i n n m n ~A """" " 1 0 - 4 micropipet. During the Process of drying the drop. the elec_I
tron microscope grid was held in a pair of tweezers. and maintained as horizontal as possible. Throughout this process, the specimen was exposed to the laboratory environment and, therefore, susceptible to contamination. However, provided that the blank sample was prepared in the same way and at the Same time ~~~~~. , .a... v*Iw fm the overall contamination could be derived. For each sample of airborn e solids, two electron microscope specimens were prepared and on(? blank for comparison. Electron Mierosca,pic Measurements. The specimens on 400mesh grids were examined in an A.E.I. EM.6G electron microscope operating at 1(M kV and fitted with a liquid nitrogen anticontamination device. The microscope was operated with the minimum electron be'am intensity necessary to give a satisfactory image without deeomlmsing the chrysotile. An init.iill ...... evaliiatin~n was made of the samples at lW0X magnification to ensure that the number distribution in the sample was satisfactory. This was followed hy selecting random grid squares and, using selected area diffraction, identifying as many fibers a8 passihle by their electron diffraction. To evaluate a sample, the svecimen stage was aliened so that a diameter of the erid could be traversed..'Starting i t one edge of the grid, fields o f k w were recorded at looox magnification, to a .grid 'quare (Figure 3) and repeated for every third grid square, giving a total of eight fields of view for each specimen. The process was repeated for the two sample specimens and the blank specimen. 810
ANALYTICAL CHEMISTRY. VOL. 45,
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results in 'l'able I clearlv show t h n t trace amounts chrysotile at the level of 0.1 pg can be t?$timated in the ~
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1971. (4) H. G. D. Williams and A. L.
Rickards, Unpublished data, 1972.
presence of large amounts of other solids. When applied to samples of airborne solids, where typically 100 mg are obtained from 1000 m3 of air, the sensitivity gives a detection limit of 0.1 ng/m3. T h e X-ray diffraction technique mentioned earlier has a detection limit of 0.1 1g/m3. A similar detection limit was claimed by Gadsen and Smith ( 5 ) for a n infrared spectrometry method when interferences from other silicates were absent. Analytical techniques based upon elemental analysis are more sensitive b u t are nonspecific for particular compounds and cannot uniquely identify chrysotile. Typical times for analysis are 5 manhours per sample, though the actual time to obtain a result is longer because of the prolonged filtration and ultrasonic procedures. The detection limit could be further improved if the chrysotile component could be concentrated by means of a separation technique. However, as a method for estimating chrysotile in urban air, the present sensitivity of 0.1 ng/m3 is probably sufficient. The values obtained from the Lancashire/Yorkshire moorland samples, which are not vastly different to those obtained from the T.B.A. factory grounds, suggest t h a t a minute amount of chrysotile exists as a general background, which is just within the detection capability of the method. The accuracy of the method has been surprisingly good. The statistical evaluation has shown t h a t the method is sufficiently sensitive to differentiate between 0.1 ng/m3 and 0.3 ng/m3. However, because of local and seasonal variations in weather and uncertainty about the precise collection efficiency of the air sampler ( I ) , estimates of ( 5 ) J A Gadsen and W L Smith A m o s Environ
4 , 667 (1970)
the chrysotile content of urban air are reliable only in terms of orders of magnitude. When a larger number of samples has been evaluated and the effects of these uncertainties are more fully understood, it may become possible to quote values in more precise terms. The continuous reference of the sample to a n equivalent, simultaneous blank is a n important feature of the method. In any laboratory interested in asbestos, there is the constant threat of serious contarnination of the samples. T o overcome this problem, sealed, disposable apparatus has been used wherever possible and the amount of handling of the samples reduced to a minimum. The method described above is specific for chrysotile. However, we are currently attempting to apply the principle of the method to amphibole asbestos where it is not easy t o define an ultimate fiber unit and, hence, the length, width, and height of each fiber need to be measured. Preliminary results from some samples containing amosite and chrysotile are extremely encouraging.
ACKNOWLEDGMENT The author wishes to thank G. F. Heron and D. V. Badami of the Research and Engineering Division, TBA Industrial Products Ltd., for their support and encouragement and the Directors of TBA Industrial Products Ltd., and the Asbestosis Research Council for permission to publish this work. Received for review September 12, 1972. Accepted November 20, 1972. T h e work was supported by a grant from the Asbestosis Research Council.
Identification of Heavier Aromatic Components in Reformed Petroleum Products by Direct Coupled Capillary Gas Chromatography-Mass Spectrometry John T. Swansiger and Frank E. Dickson Gulf Research & Development Company, Pittsburgh. Pa. 15230
In recent years, the petroleum industry has given much attention to the reformulation of gasoline blends. This interest has resulted from the necessity of reducing lead content in motor fuels while maintaining proper octane and antiknock qualities. While specific isomer and component distributions of reformed products have always been of interest as they relate to changes in refinery operating parameters, these d a t a have more recently become of major importance as a result of the major reformulations. Fortunately, techniques have been developed which can provide precise compositional information. Gas chromatography has made important contributions in providing quantitative compositional information ( I ) . However, new interest has developed in aromatic components in the range above Clo, a range where conventional GC begins to have difficulty in providing specific component identifications. Capillary gas chromatographic techniques have exL. M Taylor, C F Wantland, and I Dvoretsky A n a / 35,637-41 (1963)
( 1 ) L R Durret,
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tended the range of GC applications; but because of lack of reference compounds and peak overlap, specific identifications cannot always be made (2). The synergistic coupling of a mass spectrometer to a capillary gas chromatograph has provided a powerful technique whereby identifications of the Clo and heavier components may be accomplished. Although these heavier components do not comprise a major portion of typical reformed products, nevertheless their identification may provide significant information relating to variables in processing conditions as well as determining octane ratings of t h e final gasoline blend. Several applications of capillary GC and capillary gas chromatography-mass spectrometry (CGC/MS) to the study of petroleum light liquids have been reported. None, however, have focused their attention specifically on the Clo and heavier components. Schwartz, Mathews, ( 2 ) D. E. Willis and R . M . Engelbrecht. J. Gas Chromatogr.. 5 . 536-8 (1967)
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