than approximately 0.5u, the Shewart chart will indicate the change more rapidly, but of course changes of this magnitude are in many cases less important. Changes greater than 2 ~ r are usually apparent rapidly even to the nonstatistically minded. The technique has also been applied to the monitoring of the efficiency of a Geiger-Muller tube. This was measured initially for a long period and then daily for shorter periods using the same radioactive source. Small abrupt changes in the efficiency of the tube were observed occasionally. Other quantities, such as the variance or the range of a set of readings, could also be treated by the cumulative sum technique. This method has proved of value in two areas of radiochemistry but it is considered that the technique could be of benefit in other related fields such as x-ray fluorescence analysis or in thickness and density gauging. In addition it could be usefully applied to the routine observation of noise or background levels and instrumental efficiency of many analytical operations where large numbers of readings must be taken. Examples could be the measurement of residual current in polarography or the peak area in GC standard additions. The only fundamental difference between these applications and radioactivity measurements is that in the former cases the standard deviations must be obtained from
a series of measurements a t the start of the exercise.
ACKNOWLEDGMENT The author thanks S. Dawson for painstakingly recording so many readings.
LITERATURE CITED (1) D. A . Pantony, "Statistics, Theory of Error and Design of Experiment", R.I.C., Lecture Series No 2, London, 1961, p 24. (2) A. A. Jarrett. "Statistical Methods used in the Measurement of Radioactivity with some useful Graphs and Nomographs", AECU - 262, 1946, p 11. (3) G. Friedlander, J. W. Kennedy, and J. M. Miller, "Nuclear and Radiochemistry", John Wiley & Sons, New York, N.Y., 1964, p 176. (4) G. D. Chase and J. D. Rabinowitz, "Principles of Radioisotope Methodology", Burgess Publishing Co., Minneapolis, Minn., 1962, p 64. (5) R. Loevinger and M. Berman, Nucleonics, g (I), 26-39 (1951). (6) R. T. Overman and H. M. Clark, "Radioisotope Techniques", McGraw-Hill Book Co., Inc., New York, N.Y.. 1960, p 129. (7) M. J. Moroney, "Facts from Figures", Penguin Books, New York, N.Y., 1956, Chap. 11. (6) R. A . Faires and B. H. Parks, "Radioisotope Laboratory Techniques", George Newnes Ltd., London, 1960, p 160. (9) R. H. Woodward and P. L. Goldsmith, "Cumuhtive Sum Techniques", Oliver and Boyd, London, I.C.I. Ltd., Monograph No. 3.. 1964. (IO) W. D. Ewan and K. W. Kemp. Biometrika, 47, 363-80 (1960). (11) G. A . Barnard. J . R. Stat. SOC.,Ser. 5, 21, 239-71 (1959). (12) P. L. Goldsmith and H. Whitfield, Technometrics, 3 , 11-20 (1961). (13) W. D. Ewan, Technometrics, 5 , 1-22 (1963). (14) N. L. Johnson, J . Am. Stat. Assoc., 5 6 , 835-40 (1961).
RECEIVED for review September 21, 1976. Resubmitted July 29, 1977. Accepted July 29, 1977.
Particle Size Effects in the Determination of Respirable a-Quartz by X-ray Diffraction J. W. Edmonds" The Do w Chemical Company, Analytical Laboratories, Midland, Michigan 48640
W. W. Henslee and R. E. Guerra The Dow Chemical Company, Central Laboratory, Freeport, Texas 7754 7
Thln layers (5200 pg/cm2) of cuquartz partlcles on silver and poly(vinyl chloride) membranes are shown to exhlblt preferred orlentatlon enhancing the (1Oi1) dlffractlon line (3.34 A) for partlcles In the normally accepted size range for x-ray dlffractlon standards (C15 pm). Resplrable slllca (C5 pm) analyses performed by x-ray dlffractlon uslng a-quartz standards whose particle slre dlstrlbutlon does not match the field sample dlstrlbutlon exhlblt errors up to 300% of the amount of a-quartz present. Sample concentration In the personal samplers Is shown to be an effectlve means of Increasing sensltlvlty and establlshlng a nondestructlve analysls, but particle size determlnatlon of fleld samples Is necessary to aid selection of an approprlately sired standard. Observed threshold sensitivity for cuquartz at the 95% confidence level (20 above background) Is 11 hg/cm2.
Recognition of the health hazard due to prolonged exposure t o airborne respirable silica has prompted the development of many analytical methods for silica based upon spectrophotometric ( 1 , 2 ) ,infrared (3-5), and x-ray diffraction (6-22) techniques. For the most part, sampling procedures have 2196
ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
developed independently from the analytical methods, with the exception of modified membrane cassettes for personal samplers (10, 18). Attempts to implement one analytical method for all Dow Laboratories (23) have emphasized the desirability of a direct nondestructive technique which would permit reproducible sensitivity during interlaboratory crosschecks. X-ray diffraction was selected because of its ability to distinguish among polymorphs and its sensitivity in reported methods. The difficulties experienced in devising a suitable direct x-ray diffraction method have identified a number of problem areas which were previously unknown or inadequately investigated. Particle size distributions of field samples and quartz standards, quality of quartz standards, surface effects of sampling membranes, and modified sampling techniques are some of the critical areas to be discussed. While these results are concerned primarily with sandblasting and therefore only with a-quartz a t threshold limit values (TLV) of 100 pg/m3 of air or lower, the results should be generally applicable or identify critical problems for further investigation in other industries.
EXPERIMENTAL Many of the experiments were run independently in the Midland and Freeport laboratories but led to similar conclusions.
Therefore, the experiments will be described in general terms for clarity, and differences discussed only where significant. Standards of a-quartz examined were Minusil 5 (three lots), Minusil 15, and three standards prepared by suspending silica flour or ball milled rock quartz in ethyl alcohol or water and collecting sedimentation fractions. Relative particle size distributions were obtained using a Coulter Counter Model TAII and ammonium thiocyanate in dimethylformamide (DMF) as the electrolyte. DMF was found to adequately dissolve the poly(viny1 chloride) (PVC) membranes used for sample collection. Membranes studied included silver (25-mm diameter, 0.45 pm, Selas Flowtronics FM-25), and poly(viny1 chloride) (Mine Safety Appliances FWS-B, 5 pm, and Millipore BDWP, 0.6 pm). Membranes were mounted in the diffractometer using a brass cylinder with a concentric ring hold down. This permitted the PVC membranes to be suspended at the diffractometer center and drawn taut with no extraneous grease or other adhesive. Silver membranes were set flat on an aluminum block when possible. Many of the silver membranes exhibited a noticeable concavity which made the placement of the sample a t the instrument center difficult. All membranes were rotated in the beam in an attempt to average the effects of particle orientation and nonuniform distribution. Lack of rotation can cause intensity variations for all types of membranes and deposition methods. Membranes with known amounts of deposited a-quartz were prepared by filtration of stock suspensions or by air collection in a dust chamber. Stock suspensions were prepared by placing 0.100 g of a dried a-quartz standard in a 1-L volumetric flask and filling with deionized water which had been filtered through a 0.22-pm Millipore Duratube filter cartridge. An aliquot of this suspension (100 pg/mL) was diluted to 1 L to form a second stock suspension (2-4 pg/mL). It is critical that the suspensions be vigorously agitated for a t least 30 s before any aliquot is removed, since concentration gradients set up very quickly. All aliquots were withdrawn from the center of the suspension. A Millipore filtering apparatus was used with approximately a 16-mm effective filter diameter in the filter chimney (the exact chimney diameters were found to vary slightly). Microbalance weights (Cahn Instruments Model G or Perkin-Elmer AM-2 ratioing electrobalance) and apparent weights calculated from volume were plotted vs. x-ray intensity for a series of liquid filtered membranes. A straight line relationship was observed for x-ray intensity vs. apparent weight calculated from aliquot volume, but slightly scattered data points with occasional large discrepancies resulted when the microbalance weights were used (21). For this reason, it appears to be more valuable for liquid filtered standards to invest time in multiple determinations based solely on dilution data. A Wright dust feed mechanism (24) was used to generate a dust atmosphere in a 160-L air chamber. Membranes were placed in the filter holder cassette of a personal sampler (25) with the cyclone separator removed. Membranes were dried a t 60 "C, weighed on a microbalance, collected, dried, and reweighed. The x-ray diffractometer used was a Norelco Philips vertical goniometer fitted with a closed sample chamber, 8 compensating slit, graphite monochromator, scintillation counter, and long fine focus copper target tube. The membranes on the sample holder were mounted in the radiation chamber in a rotating sample mount. Midland scans were made a t 1/20 BO/minute from 30" to 22' 28 with the copper tube operated a t 35 kV and 25 mA. Freeport scans were made at 50 kV and 35 mA with a helium purge of the sample chamber. A standard membrane was scanned before and after the samples to correct for instrument variations, including power (kV and mA) and monochromator alignment. Increasing power to the tube does not increase the signal to noise ratio. Purging the sealed radiation chamber with helium offers about a 20% increase in signal to noise and is therefore a recommended procedure. The a-quartz 3.34-8, diffraction peak (1071) appears at 26.71' f 0.05' 20 because of variations in membrane surface smoothness created by the sample holder. For diffraction scans, the background line is drawn midway between the peak-to-peak noise from 27.5' to 25.5' 20 (Figure 1). Root mean square (rms) noise (2%) is estimated as 0.67 of peak-to-peak noise. The height of the 3.34-A line is measured above the mean background and corrected for rms noise, ( I , = I - rms/2). I , must be greater than the measured
Figure 1. (Left) 1/20 min" scan of an open faced membrane collected in the field. (Right)'/20 min-' scan of 100 pg Minusil 5 collected from
aqueous suspension (88 pg/cm*). Both membranes have 12-mm effective filter diameters. RMS noise range is estimated by the added curves at the peak bases 100
r
Min-U-Si1 5, New Lot 80
-
60
-
40
-
P e
g .g g
e
N
0
0
P,
0
:.
8 2
Diameter, Microns
100, Min-U-Si1 15, New Lot
60
20
*"
1 2
N
P
0
0
0
n m o 0
0
0
N
P 0
P
P
0
Diameter, Microns
Comparison of particle size distributions for Minusil 5 and 15. Semilog plot; (-) Coulter Counter, (,---) X-ray sedigraph
Figure 2.
rms noise (2%)to be considered observed.
Step scans were made by first manually determining the peak maximum, 28,,,, and counting for 180 s. Backgrounds were counted each for 90 s a t 28,, f 0.5'. Subtracting the two backgrounds from the counts for 28,,, gives the intensity for a-quartz, Le., I , = I - ( B , + B2).The standard deviation is calculated as % = (B E l + I + B2)'I2.2%is taken to be the detectable limit. Particle S i z e D i s t r i b u t i o n s . Procedures followed for separating a-quartz standards into different particle size distributions have been described (21). Sediments were dried and sized using the Coulter Counter. Minusil 5 and 15 (Midland lots) were sized both by sedigraphic and Coulter Counter techniques. T h e particle size ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
2197
Table I. &-QuartzResponse for Various Particle Sizes (Midland and Freeport values not normalized) a. Midland, Cu KO!, 35 k V / 2 5 mA Ag, 0.45-pm membrane Minusil 5 Minusil 15 Minusil 1 5 W
X-ray response/pg 0.3 0.8
1.0
b. Freeport, Cu Ka, 5 0 kV/35 mA, He purge Minusil 5 Standard No. 2 Millipore, 0.6-pm PVC membrane Minusil 5 Standard No. 2 MSA FWS-B. 5-um membrane Minusil 5 Standard No. 3 Standard No. 2 I
0.9 1.8
1.0 1.7
.
1.3 1.8 2.1
distributions are compared in Figure 2 . The superposition of curves (within experimental error) for Minusil5 is consistent with uniformly shaped particles, and indicates the validity of the Coulter Counter data for this sample. The lack of overlap for Minusil 15, however, is consistent with this sample having irregularly shaped particles in the larger sizes. The results possibly indicate different modes of manufacture for the two Minusils. Particle size distributions for standards No. 2 and No. 3 determined by optical and electron microscopy compared favorably to the Coulter Counter data.
DISCUSS I ON During the process of developing x-ray diffraction methods for the analysis of silica in our Midland and Freeport laboratories, prepared PVC membranes were exchanged as a check on results due to minor differences in technique and equipment (availability of helium purge, sample spinner, choice of scan speed and standard, and effective filter area). T h e apparent weights of a-quartz were calculated from the x-ray diffraction intensity of the 3.34-8. line and compared to the in-house calibration curve. The weight results for one set of membranes (Midland) were uniformly low when compared t o the other (Freeport) calibration curve. Since Minusil5 had been the standard used in Midland, and a larger sized in-house standard in Texas (21),an investigation was begun into the effects of the particle size of the a-quartz standards on the x-ray diffraction method. Silver membranes were selected because of low background in the region of the 3.34-8. line (9). From standard stock solutions (see Experimental), three membranes each were made for depositions of 20,40,80, and 100 pg of Minusil 15; Minusil5, and a fraction of Minusil 15 with the fine particles removed by suspension (“Minusil 15 W”). The relative particle size distributions for the Midland Minusil5, 15 and 15 W standards are displayed in Figure 3. Membranes were prepared in Texas according to documented procedures (21) using Minusil5 (lot differing from Midland Minusil5), and two in-house standards, No. 2 and No. 3. The relative particle size distributions are also presented in Figure 3. Depositions of standard No. 2 and Minusil 5 were also made on silver 0.45-pm membranes. The resulting x-ray diffraction intensities were averaged for each weight range for each standard, and are presented in Table I. It is clearly seen t h a t as volume averaged particle size increases, apparent sensitivity increases because of an increase in the x-ray diffraction intensity per unit weight of a-quartz. This increase in intensity per unit weight can be due to amorphous material in the smaller size standards, shadowing of small (lo0pm) on the “rough” face, but a very smooth surface under high magnification for the “smooth’ face. It has been shown that the smooth face, rather than the rough face of the MSA membrane yields much greater x-ray intensity response per unit weight of deposited silica (21),most likely because of silica not becoming entrapped deep into the membrane. The micrographs indicate that the MSA FWS-B “smooth” surface offers the best surface characteristics for analysis, and the x-ray studies confirm satisfactory intensity per unit weight. The difference between the “smooth” and “rough” faces of the MSA FWS-B membranes is apparent to the naked eye. Orientation. The Minusil 5 and 15 standards were compared by depositing 100 pg each on 12-mm circular cover glasses to eliminate membrane effects and permit the entire sample to be irradiated by the x-ray beam. The resulting x-ray intensities for the 3.34-8. line (lOIl) averaged over three independent observations under the same conditions were not equal: I M ~ ~= 0.6. ~ / Additional ~ M ~ diffraction ~ ~ ~ lines were scanned for each standard on cover glasses and for thick loadings of the bulk standards, and the relative intensities calculated (Table 111). While relative intensities of the bulk standards agree with published values, those for the 1OO-wg deposits on 12-mm cover glasses do not. Dana (26) indicates the preferred cleavage of a-quartz is ( l O T 1 ) . What is being observed is that extremely thin layers of a-quartz (no matrix) exhibit a preferred orientation favoring ( l o r l ) , the 3.34-8. d-spacing. As the deposition depth increases and gives a rougher surface, the orientation approaches random (Figure 5). Using relative intensities as indicators (Table 111), it can
+-‘
2
W
a
I
25t t
20
-i
Midland Min-U-Si1 5
I
Texas Min-U-Si1 5
I
1‘00
80
/
60
40 20
+-’
25
100
a“
20
80
-
f
15
60
p
10
40
5
20
% L W
E 3 0
0
+-’
100
C
1)
2W
80
a
69
40 E
20
3
Diameter, Microns
Diameter, Microns
Comparison of Coulter Counter derived particle size distributions for a-quartz standards. Bar graph indicates volume percent for each data channel, plotted as equivalent diameter, pm. Line graph represents cumulative volume percent
Flgure 3.
be seen t h a t preferred orientation is apparent on the MSA FWS-B membrane as well as the silver membrane. I t appears that two phenomena account for the decreased x-ray intensity response per unit weight on silver membranes. First, the smaller particles are “shadowed” (hidden from the x-ray beam) by the rougher silver membrane surface and, second, the rougher surface gives a more random orientation to the deposited a-quartz particles, Le., preferred orientation enhances the ( l O T 1 ) diffraction line on the flat PVC membranes. This effect can be used to enhance the threshold sensitivity of an analytical method, however, if it is recognized. The observed effect of a-quartz particle size on the calibration curves is therefore primarily due to preferred orientation. If a standard is used whose particle size is significantly larger than the particle size of the field samples,
then a larger x-ray intensity per unit weight of a-quartz will be observed, a lower threshold sensitivity deduced, and the actual amount of a-quartz on a field sample will be underestimated. For example, t h e error can approach a factor o f three as exhibited by the different responses of Minusil5 and 15 (Table 0. Standards. The obvious standard t o use is one which matches a “respirable” size distribution obtained by the sampling apparatus (27). Since this is not generally available, the best approximation is Minusil5 or any a-quartz standard whose particle size distribution fits the field sample distributions. Unfortunately, a-quartz standards, however similar in particle size, are not identical. The pulverizing process creates an amorphous surface on the a-quartz particles approximately 0.03 pm thick (28-35), making in-house standard ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
2199
Table IV. Relative X-ray Intensities for Various Sources of quartz"
Quartz t y p e Minusil 5 (new lot) Minusil 5 (old lot) Minusil 1 5 (new lot) Minusil 1 5 (old lot) Minusil 15W cut No. 1 Ball milled rock quartz
Volume X-ray averaged response/ diameter, pm pg 1.7 3.8
1.2 1.1
4.5
1.5 1.7 1.9 1.4
7.2 1.7
4.3
' 100 p g deposited on 12-mm cover glass slips.
Flgure 4. Electron micrograph of Minusil 5 particle deposited from liquid suspension on silver 0.45-pm membrane. The irreguhrly shaped particle would preferentially orient on a smooth surface, and be "shadowed" on a rough surface. Marker indicates 1 p m . (Adapted with permission from (27)) 7
0.16 I
t 0
I
I
4ca
2w
1
1
I
MM
800
lo00
1200
pq Minus81 15W
Figure 5. a-Quartz x-ray intensity ratio, I ~ ~ ~ ~ compared / I ~ l ~ to j ~ weight l , of Minusil 15W deposited on 12-mm cover glass slips
Table 111. l / I , l o ;
1)
@-QuartzPreferred Orientation
(ioio)
(1120) 2.458
(1122) 1.817
0.11
0.21
0.11 ... Bulk 0.18 0.11 Minusil 5 500 p g an Ag, 0.45 pm 0.18 0.05 Bulk 0.16 0.08 Minusil 15, air collected, MSA FWS-B 260 p g 0.17 0.09 Bulk 0.15 0.09 Minusil 1 5 W on coverglass No. 1, 100 p g 0.08 0.02 No. 2, 1 0 0 p g 0.10 0.06 Bulk 0.15 0.09 Minusil 5, 3.5% in Cabosil Bulk 0.16 0.08
0.10 0.21
(hkil)
cl-spacing, A
4.26
Literature 0.18-0.22 Ball milled rock quartz 100 p g on
12-mm
coverglass
2200
0.09
0.17 0.09
0.17 0.03 0.07 0.17
0.19
ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
Volume Average Diameter pm
Flgure 6. Relative x-ray intensities of a-quartz (107 1) for particle size fractions of Minusil 15, 100 wg on 12-mm cover glass slips. Upper curve is theoretical fall off of x-ray intensity assuming a 0 . 0 3 - j m amorphous shell and spherical particles in the particle size distribution generation inadvisable. For example, pure rock quartz ball milled, sized, and scanned by x-ray diffraction is compared to commercial Minusil standards in Table IV. Treatment with 10% HF to remove the amorphous layer (28) is not advised, as the field samples cannot be similarly treated without complicated manipulations detrimental to a fast, reliable, direct analytical method. T h e physiological effects of this amorphous layer are still unclear. T h e best solution appears to be the use of a universally available a-quartz standard which has a well documented particle size distribution below 5 pm. This would then permit interlaboratory exchange of membranes for verification purposes, and aid in making silica exposure data uniform for future health and environmental comparisons. One remaining problem, however, is that there exists no direct technique for determining the percent crystalline a-quartz in an x-ray diffraction standard other than x-ray diffraction. The work necessary to determine the quality of a n a-quartz x-ray diffraction standard is sufficient t o justify certification and availability through a single source. The effect that the amorphous nature of the surface of the a-quartz particles in field samples will have on x-ray diffraction response is still unknown. Sandblasting may have an entirely different effect on particle surface than, for instance, foundry activities or quarrying. Errors may remain in the estimation of the amount of a-quartz from various sources, even when compared to a universal standard. I t is not felt that these errors are large in the sandblasting field samples observed, where only 3% of the particles passing the cyclone are smaller than 0.4 Nm and therefore have a negligible amorphous content. T h e amorphous content present in Minusil 15 was investigated by sedigraphically separating various particle size ranges (see Experimental) and measuring x-ray intensities for three depositions of 100 wg of each fraction on 12-mm cover glasses. T h e results are displayed in Figure 6. Assuming a 0.03-pm shell of amorphous silica surrounds each a-quartz particle (28), a curve of x-ray intensity vs. volume average
Table V. Relative X-ray Intensities for &-Quartz(1071) for Air and Filtered Liquid Suspensions of Minusil 5 Collected on MSA FWS-B 5-bm Membranes Using a 12-mm Effective Filter Diameter X-ray responselfig Air filtered Liquid filtered
Figure 7. Exploded view of a Bendix 25-mm filter cassette and cyclone. Also shown are the Teflon pre-membrane insert and the 8-mil stainless steel post-membrane insert
diameter can be calculated using the particle size distribution for each of the three Minusill5 sedigraphic cuts and assuming random orientation. These calculated points are normalized and also displayed in Figure 6. I t can be seen that the observed x-ray intensity (with preferred orientation) falls off faster than expected for the smaller particle fractions of Minusil 15. While the fall off may be due in part to the loss of enhancement due t o preferred orientation, the x-ray intensity should not fall to nearly zero with the smallest fraction. This indicates there is a significant fraction of noncrystalline material in the smaller particle sizes. While this size fraction may be negligible compared to the sample bulk, the percent crystalline silica is not 100% as presumed, but unknown. I t is also indeterminable without critical assumptions. The lack of standard characterization in published methods (1-58-20, 22) therefore makes it impossible to make a fair comparison of the sensitivities or detection limits of these methods. S a m p l i n g . The two-stage respirable dust separator has achieved acceptance as a sampling device. The variations in available filter holders have forced most analytical methods development to deal with the collected membrane, with two exceptions (10, 18). It is obvious that if the x-ray or infrared beam cannot irradiate all of the deposited material, one must assume uniformity of deposition or redeposit the sample in order to perform the analysis. Since redeposition techniques are time consuming and prone to introduce additional error, it seems preferable to confine redeposition to cases where severe interferences must be removed. It therefore appeared more reasonable to modify the existing cassettes than to design a separate filter holder as had been done previously (10). A Bendix 25-mm filter cassette and cyclone (251, Figure 7, were selected since the collection membrane would fit directly onto the diffractometer sample spinner attachment. This filter holder had been designed in conjunction with Gebhardt (18), but still retained an effective filter diameter of 21 mm, substantially greater than both the
0 . 9 ? 0.1 1.0 i 0.03
x-ray beam (12.5 mm square) or the liquid filtration chimney used for standard membrane preparation (- 16-mm diameter). The first modification was a Teflon insert placed between the cyclone outlet and the collection membrane (Figure 7 ) . The insert had a 15.5-mm cylindrical hole, so that deposition would cover the same effective filter area as the standard membranes from liquid filtration. a-Quartz dust was deposited on membranes in an air chamber as described under Experimental, but resulted in a nonuniform dust distribution. Since the open chamber behind the membrane and the Teflon insert appeared to control deposition, a post-membrane disk insert was fabricated from 8-mil stainless steel, 25 mm in diameter. A 12-mm hole was punched concentrically so t h a t air flow would be forced to the membrane center and all material would be deposited in an area which would be completely irradiated by the x-ray beam. Placing the insert behind the membrane does not give a membrane-insert interface for a-quartz entrapment. Air expansion and convection into the chamber behind the insert might affect the uniformity of deposition, but all deposited material would be irradiated regardless. Air chamber experiments were repeated using the postmembrane insert and Minusil5. Membranes were treated as above and the results are displayed in Table V. The intensity curve for the air deposited membranes is quite linear for values less than 100 pg/membrane, the region of interest, and the calibration curve for filtered liquid suspensions of Minusil 5 is seen to be the same within experimental error. This is an indication that liquid filtered standard membranes can be used for analysis, thus avoiding the complications of dust feed mechanisms and air chamber equilibration. Inserts made for the more common 37-mm cassettes have proved just as effective in field use. M a t r i x Effects. Spurious effects of iron contaminants might be expected with fluorescence because of the Cu radiation and absorption. No adverse effects were observed for the x-ray intensity of a-quartz when admixed with a 1:l mixture of FesOBand Fe304in a 1:4 weight ratio with a-quartz. The data points for this mixture were observed to fall on the a-quartz calibration curve (21). Similar results were obtained by Bradley for iron oxides ( 8 )and Gebhardt with calcite and feldspar (up to 80% total sample weight) (18). Field Samples a n d Cyclone Operation. Field sampling was made with the post-membrane insert in a two-stage cyclone separator (25). Flow rate checks with a membrane and insert in place indicated a 1.7 to 2.0 L/min. flow could easily be maintained for the MSA FWS-B 5-pm membrane, but a maximum of only 1.6 L/min for the Millipore BD 0.6-pm membrane. Previous studies indicate that the IO-mm cyclone passes a particle fraction in agreement with the Los Alamos curve (27)and that this separation efficiency does not change in the flow rate range of 1.7 to 2.1 L/min (36). Field samples were collected both the respirable zone under the sandblasters hood using the cyclone separator and in the open air adjacent to the sandblasting operation using an open faced membrane. Typical particle size distributions with and without the cyclone are displayed in Figure 8. T h e size selected material is seen to have a size distribution similar only to the Minusil 5 standard. The cyclone is also seen to be quite efficient a t the 2.0 L/min flow rate. An x-ray difPiNALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
2201
Table VI. For Scanned Membranes, 01- Quartz Threshold Detection LimitIMembrane at 9 5 % Confidence Level, Imin, 28 scan Cu KCY3 5 k V / 2 5 mA, Threshold Membrane Ag, 0 . 4 5 Mm MSA FWS-B,
Minusil Minusil Minusil Minusil Minusil Minusil Minusil
Condi tionsa 5, 1 5 . 5 mm, liquid 15, 1 5 . 5 mm, liquid 15W, 1 5 . 5 mm, liquid 5, 1 5 . 5 mm, liquid 15W, 1 5 . 5 mm, liquid 5, 1 2 mm, liquid 5, 1 2 mm, air
(20 1,
25
I
Field Sample No Cyclone
100 c
C
:20
80
2
e
h
Mg
25 11 7 15 6 11 11
I
a
15
60
10
40
5
20
-
3
a a-Quartz standard, effective filter diameter, filter mode.
Table VII. Increased Sensitivity for Three-Point Step Scans, 1 0 0 p g Minusil 5 on MSA FWS-B, Liquid Filtered Standard
4-
%
Time, B, P
B2
S
Count
10 20 10 30 60 30 90 180 90 180 360 180
103 450 118 311 1362 342 942 4012 997 1919 8207 2006
I=PB , - B2 229
20 = 2 (P f Bl +
relative error, Threshold detection limit,
B2)1’2 MP 52
23
709
90
13
2073
154
7
4282
220
5
a In this example only, % relative error = threshold detection limit at 95% confidence level.
fraction scan of an open-faced sample indicates the signal to noise ratio encountered and the low level of interferences (Figure 1). Detection Limits. The variation of threshold detection limit with particle size distribution for scanned membranes is summarized in Table VI. Table VI1 summarizes the increased sensitivity gained in using a three-point step scan method, in which considerable time is saved and fewer errors induced.
CONCLUSIONS 1. T h e tendency for a-quartz to exhibit preferred orientation in thin deposited layers on smooth surfaces is responsible for the increased x-ray diffraction intensity per unit weight on PVC membranes. The rougher surface of silver membranes tends not only to give a more random particle orientation, but also to “shadow” smaller particles from the x-ray beam, both resulting in lower unit intensity response than observed for smooth PVC membranes. This orientation effect can be used to enhance a-quartz threshold detectability on smooth PVC membranes compared to t h a t on silver membranes. Rotation of the sample in the beam is also advisable to even out the effects of this preferred orientation. 2. The tendency to select a “pure a-quartz” standard for use in respirable silica analysis without regard for particle size distribution will lead t o spurious, although internally con2202 * ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
I
Field Sample With Cyclone 25
-
20
2 L
w
E
15
-
8
-
10
5
in b ’ m o o w o a
,-
9
.-
,-
,N
N VI
m
~
0
I”
w
y l -
o
~
-
A - d N N W -e P ,m ,m 0 ) P N ? P i.” N P 0 --eo w 0 0 w 0 0 m o ~ ~ o m o N o
W C
2
80
2
s
W
-
60
8 40 20
m
o
o
Diameter, Microns
Figure 8. Comparison of Coulter Counter derived particle size distributions for field collected material with and without the 10-mm cyclone separator. Material with diameter greater than 5 p m in the lower graph is particle/membrane agglomeration
sistent, results. For instance, maximizing threshold detection by selecting Minusil 15 over Minusil 5 can lead to almost a threefold underestimation of the amount of respirable silica on field samples collected with a cyclone size selector. Therefore, an a-quartz standard with a particle size distribution nearly 100% in the “respirable” range should be characterized and be made universally available even though this results in an apparent loss of “sensitivity” above background on selected membranes. 3. Simple modification of personal samplers (25- and 37-mm) to permit collection of dusts in an area (e.g., 12-mm diameter) t h a t is entirely irradiated by an x-ray or infrared beam does not decrease the efficiency of the sampler, but will concentrate the sample for more sensitive and rapid analysis. The samplers (10-mm cyclones) can efficiently operate at flow rates from 1.7 to 2.0 L/min. 4. The availability of a “respirable” a-quartz standard and the deposition of a-quartz in an area completely irradiated would make round-robin sample exchanges a convenient reality, and provide a meaningful basis for the reported exposures to respirable silica dust. 5. The fall-off of calibration curves a t 100 pg/cm2 is most likely due not to absorption but to the loss of preferred orientation favoring the 3.34-A d-spacing of a-quartz. The deposited layer is thick enough to favor more random orientation so that the observed x-ray intensity of the (lOT1) peak begins to decrease to the expected relative intensity value for a randomly oriented bulk sample. 6. Determination of the particle size distribution of selected field samples is necessary to ensure the validity of the standard used.
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ACKNOWLEDGMENT T h e authors thank G. R. McGowan and B. W. Pynnonen for the particle size analyses, R. E. Cook for aid in field sampling, H. 0. Yakel for aid with the dust chamber work, L. R. Ruhberg for help with the initial x-ray studies, L. D. Martin, M. R. McCullogh, and D. W. Krieg for studies on the scanning microscope' D' c' Benefiel On the transmission electron microscope, a n d R. R. Langer and B. S. Horvrath for discussions a n d support.
LITERATURE CITED N. A. Taivitie, A m . Ind. Hyg. Assoc. J . , 25, 169-178 (1964). D. V. Sweet, F. R. Wolowicz, and J. V. Crable, A m . Ind. Hyg. Assoc. J . . 34, 500-506 (1973). (3) J. W. Cares, A. S. Goidin, J. J. Lynch, and W. A. Burgess, A m . Ind. Hyg, Assoc. J . , 34, 298-305 (1973). (4) D. J. Larsen. L. J. von Doenhoff, and J. V. Crable, A m . Ind. Hyg. Assoc. J . . 33, 367-372 (1972). S.2. Toma and S. A. Goldberg, Anal. Chem., 44, 431-432 (1972). G. L. Clark and D. H. Reynolds, Ind. Eng. Chem., Anal. Ed., 8, 36-40 (1936). H. P. Klug, Anal. Chem., 25, 704-708 (1953). A. A. Bradley, J . Sci. Instrum.. 44, 287-288 (1967). J. Leroux and C. A. Powers, Staub-Reinhak. Luft(€ng. Ed.), 29, 26-31 (1969). J. Leroux, A. B. Davey, and A. Paillard, Staub-Reinhalt. Luft(Eng. Ed.), 32, 27-31 (1972). J. Leroux. A. B. Davev. and A. Paillard. A m . Ind. Hva. Assoc. J . . 34. 409-417 (1973). G. Knight, W. Stefanich, and G Ireland, A m . Ind. Hyg ASSOC J . , 33, 469-475 (1972). G. Knight, R. Kowalchuk, and R. Yourt, A m . Ind. Hyg. Assoc. J . . 35, 671-680 (1974). G. C. Allen, B. Samimi, M. Ziskind, and H. Weill, Am. Ind. Hyg. Assoc. J . , 35, 711-717 (1974). H. E. Bumsted, A m . Ind. Hyg. Assoc. J . , 34, 150-158 (1973). R. W. Freedman, S. 2 . Toma, and H. W. Lang, A m . Ind. Hyg, Assoc. J . , 35, 411-418 (1974).
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(17) A. Mangia, Anal. Chem., 47, 927-929 (1975). (18) R. F. Gebhardt, Martin Marietta Cement Technical Center, Baltimore, Md. Paper presented to the September 1975 meeting of the Portland Cement Association at Tucson, Ariz. (19) M. T. Crosby and P. S. Hamer, Ann. Occup. Hyg., 14, 65-70 (197'1). (20) S. Altree-Williams, Anal. Chem., 49, 429-432 (1977). (21) W. W. Henslee and R. E. Guerra, "Direct Quantitative Determination of Silica by X-ray Diffraction on PVC Membrane Filters", Advan. X-ray Anal., 20, 139-152, 1977. (22) E. T. Peters, "Evaluation of the NIOSH X-ray Diffraction Method for the Determination of Free Silica in Respirable Dust", Contract No. CDC99-74-51 ., NIOSH, 4676 Columbia Pkwy., Cincinnati, Ohio 45226, May
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(23) "Occupational Exposure to Crystalline Silica, Criteria for a Recommended Standard", HEW publication No. (NIOSH) 75-120, 1974. (24) B. M. Wright, J . Sci. Instrum., 27, 12-15 (1950). (25) Bendix Environmental Systems, Model No. 2419107. (26) E. S. Dana, "A System of Mineralogy". 6th ed., J. Wiiey & Sons, New York, N.Y., 1937. (27) Aerosol Technology Committee, American Industrial liygiene Association, Am. Ind. Hyg, Assoc. J . , 31, 133-137 (1970). (28) . . G. Naoelschmidt. R. L. Gordon. and 0. G. Griffin. Nature(London). 169 (43ooj, 539-540 (1952). (29) P. B. DemDster and P. D. Ritchie, Nature(London),169 (43001, 538-539 (1952). (30) D. W. Cblland, W. M. Cummirg. and P. D. Richi, J. Appl. Chem. (Lorobn), 2, 31-41 (1952). (31) R. L. Gordon and G. W. Harris, Nature(London), 175, (4469), 1135 (1955). (32) C. M. Jephcott and H. F. V. Wall, AMA Arch. Ind. Health, 11, 425-430 (1955). (33) G. Nagelschmidt, Analyst (London),81, 210-219 (1956). (34) G. W. Brindley and S. Udagawa, J. A m . Ceram. SOC.,42 (12), 643-644 (1959). (35) R. W . Grimshaw, "The Chemistry and Physics of Clays and Allied Ceramic Materials", 4th ed.. Wiley-Interscience, New York, N.Y., 1971, p 708. (36) G. R. McGawan, Dow Chemical Co., Midland, Mich., private communication, 1975.
RECEIVED for review April 18,1977. Accepted August 31,1977.
Proton Induced X-ray Emission Analysis of L-Cells Grown in Vitro R. R. Zombola,* P. A. Kitos, and
R. C. Bearse
University of Kansas, Lawrence, Kansas 66045
The trace element concentrations of Lceils grown in vltro have been determined by PIXE analysis. The preclslon of the technique was determined by comparing measurements of 20 targets made from a common sample. Nineteen separate cultures were analyzed to determine the variability of "identically" grown cells. The results from this latter experiment indicate that the elemental concentration in ppm of each element in L-celis is K (13033 f 649), Ca (74.7 f 10.7), Mn (5.0 f 0.5), Fe (101 f 3), Cu (12.3 f l o o ) ,Zn 57.6 f 2.8). The system was shown to be linear for variations in elemental concentration.
Proton induced x-ray emission (PIXE) has been recognized as a useful method for determining the elemental composition of small samples ( I ) . A major advantage of this technique is t h a t a sample can be analyzed quickly, and all elements between C1 and M o are detected simultaneously if they are present in large enough quantity. The purpose of this paper is t o report the details involved in applying this technique to the elemental analysis of cell cultures. These cell samples ape, by their nature, small in total mass and contain several el-
ements a t concentration levels amenable to measurement by PIXE. T h e cells used here, L-cells, are derived from a line of fibroblastoid mouse cells. Comparison of elemental concentrations of these L-cells to L-cells in various pathological states may be helpful in understanding the nature of these states. There is also some evidence that healthy and cancerous cells have different trace element concentrations (2). Thus, application of this technique could open some interesting avenues of research in cell biology.
EXPERIMENTAL The cells used were NCTC clone 929, strain L, mouse cells which have been in continuous culture at the University of Kansas for 12 years. Growth takes place on the interior walls of glass 250-mL screw-cap milk dilution vessels, each of which contains 35 mL of a modified Waymouth's MD 705/1 nutrient medium (3). The cells attach themselves t o the glass shortly after inoculation and remain there during incubation at 37 OC. After 7 aays, the growth medium is removed, the cells are rinsed with salihe solution, and 5 mL of deionized-distilled water is pipetted into each vessel to lyse the cell walls. The cells are scraped from the sides of each bottle and suspended in the distilled water. Each cell suspension is decanted into a plastic 50-mL centrifuge tube, lyopholized, and transferred into a 1-mL Pyrex beaker. After ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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