2 149
Anal. Chem. 1981, 53. 2149-2152
-~
Table IV. Effect of Various Solvents on Cerium Analysis (10%v/v on 20 ppm of Cerium) re1 net exptl vs. solutions intens actual value, % distilled watera HNO, HC1 "30, H,PO, H,SO,
81 68 68 66 55 58
100
",OH
27 77
33
ethanol a
84 84 81 68
72 95
Standard solution.
major disadvantage of ICP over FAA. Several different solvents were used in this work for determining the degree of thle transport interference. The results are shown in Table IV. The data suggest that the solvent composition of the sample should be matched as closely as possible to that of reference solutions. Nebulizer Effects. T'hree of the most popular nebulizers, stainless steel, cross-flow, and concentric glass nebulizer, were compared for their capability of efficient aerosol generation. By optimization of the operational conditions for each nebulizer, all three devices produced similar detection limits, with stainless steel being the best. The stainless steel nebulizer was used in the paint analysis because this device can tolerate a relatively higher salt content in the sample. The other two nebulizers were clogged partially or completely a few minutes
after starting the analysis. These nebulizers require frequent cleaning to restore the original flow rate of the aerosol and necessitate restandardization of the ICP. The present study establishes a relatively simple and rapiid analytical procedure for determining the ultratrace amount of cerium in paint samples by using ICP-OES. The determined paint concentration of Ce was 0.032% 0.001 which is within the range of the manufacturer's specification. The data indicate good agreement for both direct determinatioln and standard addition methods. The calibration curve extended over a broad smalytical dynamic range for Ce (0.2-700 ppm). This implies that both macroamounts and ultratrace amounts of Ce can be analyzed for in a single reference solution.
*
LITERATURE CITED (1) Boumans, P. W. J. M.;de Boer, F. J. Spectrochim. Acta, Part B 1975, 308 309-340. (2) Butler, Constance C.; Knlseley, R. N.; Fassel, V. A. Anal. Chern. 1975, 47, 825-829. (3) Kniseley, Richard N.; Butler, Constance C.; Fassel, V. A. Anal. Chern. 1969. 41. 1494-1496. (4) Thomas, P. E. "Resonance Lines", Cary Instruments: Monrovia, CA, Vol. 1 (I), p 6. (5) Yoe, J. H.; Koch, H. J. "Trace Analysis", 1st ed.; Wlley: New York, 1957; p 626. (6) Roboz, J. "Introduction to Mass Spectrometry, Instrumentation and Technlques", 1st ed.; Wlley: New York, 1988; p 390. (7) "Seml-Quantitative Spectrochemical Analysis"; Spex Industries, Ino.: Metuchan. NJ. 1964. (8) Larson, G: F.;'Fassel, V. A.; Scott, R. H.; Kniseley, R. N. Anal. Chem. 1975, 47, 238-243
RECEIVED for review April 24, 1981. Accepted July 8, 1981.
Polyatornic Interferences in High-Resolution Secondary Ion Mass Spectra of Biological Tissues Margaret S. Burns Depatfments of Ophthalmology and Biochemistry, Albeti Einstein College of Medlcine/Montefiore Hosptial and Medical Center, 11 1 East 210th St., Bronx, New York 10467
Formation of polyatomic species during ion bombardment is a complicating factor in secondary ion mass spectrometric analysis of materials (1,2).Biological specimens are almost entirely C, H, N, and 0 and it is expected that there will be many hydrocarbon polyatomic species which can interfere with quantitative determination of elements of interest in physiological experiments ( 3 , 4 ) . In the present work high mass resolution spectra of tissue preparations are examined to assess the importance of these potential interferences.
EXPERIMENTAL SECTION Eyes were enucleated from cats, anesthetized with Nembutal, or from toads subsequent to guillotining. The eyes were quickfrozen in an isoperitane slush cooled in liquid nitrogen and lyophilized by use of a chemisorption vacuum system (5). The samples weire held at -30 "C until dry. Samples of retina and choroid a few millimeters square were cut and directly embedded in Spurr's law-viscosityresin. Ten micrometer sections were cut with a dry glass knife, pressure mounted on a boron-doped silicon wafer (Monwanto, Inc., Dedham, MA) and overcoated with 500 A of 99.99% Au to provide conductivity. The Cameca IMS-3F (Charles Evans and Associates, San Mateo, CA) was operated with either positively charged cesium primary ion beam or with positively charged oxygen. Negative secondary ions were collected under cesium bombardment and positive ions with oxygen. Oxygen bombardment of 5.5 keV was used with a 250 pm X 250 /bm raster and 500 nA beam current. 0003-2700/81/0353-2149$01.25/0
High mass resolution spectra of selected areas were taken by use of a 50 pm diameter field aperture. Cesium bombardment was at 14.5 keV and 100 nALbeam current and the same geometry as with oxygen.
RESULTS AND DISCUSSION The type of biological tissue examined in this study is not crucial because we are examining ions that are prevalent a t high concentrations in all tissues. The retina and choroid provide a convenient preparation because they consist of different anatomical structures with known differences in chemical composition (Figure 1). For example, the nuclear areas are largely deoxyribonucleic acid and have a higher I' content than other cell structures. It has been shown that the choroid and pigmented structures are rich in calcium intensity (6). With oxygen bombardment the positive secondary ions arle predominantly the alkaline elements (Figure 2). The high mass resolution spectrum at 23+, 24') 35+, 39+, and 40' from the region of cat choroid is typical of the spectra obtained from all tiseue areas (Figure 3). Sodium, 23') i13 always a single peak. Magnesium, 24+, is always a doublet and sometimes contaiins a poorly resolved shoulder on the C2 peak. This is probably NaH'. A mass resolution of 10000 is necessary to separate these signals at 10% valley definition (Table I). The relative height of Mg+ to Cz+varies depending 0 1981 American Chemical Society
2150
ANALYTICAL CHEMISTRY, VOL. 53, NO. 13, NOVEMBER 1981
n I
z
I D Figure 1. Schematic diagram of retina and choroid: BV = blood vessel; M = melanocytes; RPE = retinal pigment epithelium; ROS = rod outer segment; RIS = rod inner segment; ONL = outer nuclear laycr; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GC = ganglion cell; NFL = nerve fiber layer.
40
m le
Figure 5. Mass 40' of INL and IPL region of cat retina.
lob[ 105
02'
N *.
CO'
K'
L,
-
C2*
10'10'
C20'
24
39
40
mI s
m le
Figure 2. Low mass resolution spectrum of cat choroid using 02+ bombardment.
Figure 6. Spectrum of selected ions in epoxy resin to which calcium was added.
m le
23
24
35
39
40
mle
Figure 3. High mass resolution spectrum of selected ions from cat choroid using 02+bombardment.
i061
02'
105
b
24
C
24
24
m /e
Figure 4. Mass 24' from different specimens: a = IPL of toad retina: b = choroid of toad retina; c = epoxy resin. upon the tissue area (Figure 4). Mass 35+ is a doublet and consists of Cl', arising from both resin and tissue and NaC+. The potassium peak, 39+, is generally lo4 more intense than the background. One to three small peaks are always present with K+ and are NaO' and C3H3+. The calculated mass resolution is 5750 at mass 39' by 10% valley definition. The second peak is calculated to be separated by 0.02 amu from K+ and is most likely NaO+ which is 0.02098 amu from K+.
Figure 7. Low mass resolution spectrum of cat choroid ulsng Cs' bombardment. Similarly the fourth small peak is 0.064 m u from K+ and is probably C3H3+. These peaks occur in all tissues examined and have less than intensity of K+. Calcium, 40+, is consistently one major peak at 1000 to 10 000 counts/s. There are one to three peaks with less than 10 counts/s. On the basis of their separation from Ca+ they are most likely MgO+, CzO+and C3H4+.In some areas a signal that corresponds to 3gKH+was seen (Figure 5). Separation of these two peaks requires only 4500 mass resolving power, well within the instrumental operating conditions (Table I). In epoxy resin containing added calcium, only hydrocarbon species are seen along with a small potassium signal (Figure 6). Cesium bombardment of tissue or resin gives a low mass resolution spectrum with numerous polyatomic species present, presumably involving carbon atoms (Figure 7). The high mass resolution spectra were consistent in both cat and toad retinal areas and a typical example is shown in Figure 8. Mass 31- is resolved into P- and CH30- with at least 10' greater intensity in P-. Mass 32- is predominantly S- with an intensity of lo6 counts/s and the PH- and 0, interferences are about lo2 counts/s. Mass 35- is predominantly C1- with a minor interference that is most likely NaC-. Since tissue specimens are heterogeneous in chemistry and morphology, one cannot assume the above conclusions will
ANALYTICAL CHEMISTRY, VOL. 53, NO. 13, NOVEMBER 1981
21111
Table I. ]Required Mass Resolving Power amu nominal mass species 23Na "BC
23
22.98978 22.00931 23.01 091
,7~io a4Mg
24
23.98 504 23.99760 24 .OOOOO
23NaH 'T, a6Mg "CN IaC,H, P 30SiH CF CH30
26
3 1.
25.98259 26.00307 26.01 565 30.97376 30.98158 30.99840 31.01839
32s
3 2!
31.97 207 31.98158 31.98983
)'PH 1602
35
34.96885 34.98978
3 5 c 1
a3NaC
39K
39
38.96371 38.98469 39.02347
NaO C3H3 40Ca KH
40 1099{
39.96259 39.97 153 39.99491 40.03130
c";;, Cr
52
51.94051 51.99492 52.03128 54.93 805 54.95862 54.97960 55.05474
c30
C4H4 Mn
55
3 9 ~ 0
NaO, C,H,
Fe
56
55.93494 55.95750 56.06256 57.93535 57.95863 58.07820
40ca0 C4H8 58Ni
58
Na3T1 C4HIO e3cu C5H3 75A~ 40CaCI C,H,
63 75
62.92959 63.02346 74.92171 74.93114 75.02346
1 t t
1 tt
t 1 I 1 i tt 1
1089
1
1177
1603
{
1910
786
1
1269
694
}
1257
1800
32
3362
}
1856
10000
1
2067
1 1\ 3961 1842 842 1
1551
1669 652
1006
582
}
1236
57 2
1
955
47 1
}
1322
438
}
2479
406
}
2489
1
7945
267 1
670 736
105
31
1
t
35
m/e
Flgure 8. High mass resolution spectrum of 31-, 32-, and 35- using
Cs' bombardment of cat choroid.
be valid for every specimen examined. However, because the concentrations of thse elements are both high and relatively similar in different tissues, one may expect that the findings will be qualitatively similar. Moreover, the retinal tissue used for this study is variable in terms of cell type, cell origin, intraand extracellular space, and morphology and the results were reproducibly found in all tissue areas from two different species, indicating the generality of the findings. The relative
peak intensities of the mass resolved species were similar whein the same area or cell type was repeatedly analyzed, indicating that qualitative reproducibility is reasonable and local charging is not a problem. Therefore, quantitation can be done at low mass resolutions without significant error due to mass interferences (4). Each individual tissue must be routinely examined for these parameters before exhaustive studies of elemental localization and quantitation are done. This is especially true for trace elements. The problem of interferences will become more severe as the concentration of the element decreases (7). Calculations of the resolving power necessary for a few trace elements of interest are presented in Table I. Although the resolving power required is not great for separation of the peaks, the intensity of the hydrocarbon or Na and K containing species will be consistently high. Thus, one must bo sure to use adequate imass resolution to demonstrate an interference if present.
ACKNOWLEDGMENT The assistance of and discussions with Vaughn Deline and technical assistance by Judith Channer are gratefully acknowledged.
2152
Anal. Chem. 1981, 53, 2152-2154
LITERATURE CITED (1) Slodzlan, G. Surf. Sci. 1975, 48, 161. (2) Bakale, D. K.; Colby, B. N.; Evans, C. A., Jr. Anal. Chem. 1975, 4 7 , 1532. (3) Burns-Bellhorn, M. S. I n “Microbeam Analysis in Biology”; Lechene, c., Warner, R. R., Eds.; Academic Press: New York, 1979; p 129. (4) Burns-Bellhorn, M. S.; Flle, D. M. Anal. Blochem. 1979, 92,213-221. ( 5 ) Stumpf, W. F.; Roth, L. J. J . Hlstochem. Cytochem. 1987. 15, 243-251. (6) Belihorn, M. 8.; Lewis, R. K. f x p . €ye Res. 1976, 22, 505-518.
(7) Truchet, M.; Vovelle, J. Calcif. Tissue Res. 1977, 34, 231.
RECEIVED for review November 3, 1980. Resubmitted July 10, 1981. Accepted July 10,1981. This study was supported in Part by NIH Grant No. EY 02903, A Research Career Development Award (EY00104), an unrestricted grant from Research to Prevent Blindness, New York, and the National Society for the Prevention of Blindness, New kiork.
Carbon Dispersed in Glass Fibers as an Adsorbent for Contaminant Enrichment and Fractionation Lawrence M. Smith Columbia National Fisheries Research Laboratory, U.S. Fish and Wildlife Service, Rt. 1, Columbia, Missouri 6520 I
Activated carbon, the age-old purifier, continues to offer expanded applications in the treatment of municipal and industrial wastes, in the recovery and purification of synthetic and natural substances, and in numerous other chemical and environmental processes (1-3). New applications of activated carbons in the separation and purification of organic compounds are largely the result of the availability of more sophisticated preparations of activated carbons possessing more refined chemical and physical properties (3-7). Research at the Columbia National Fisheries Research Laboratory has shown that the application of the uniquely selective adsorptive properties of certain activated carbons to the purification of organic compounds can be seriously limited by the development of high back-pressure in chromatograply columns packed with finely divided carbon and by poor recoveries of strongly absorbed substances, even from sub-gram quantities of carbon. The adsorptive properties of some specially prepared carbons were determined to be particularly well suited for the separation of several important classes of toxic compounds and numerous isomers within these classes (8). The difficulties in applying these unique properties to trace analysis were primarily associated with the physical preparation of the adsorbent in a chromatography column; effective recovery of the desired compounds often required the use of less than gram quantities of powdered activated carbon. Subsequent research a t the Columbia laboratory led to the development of dispersed carbon adsorbents with physical properties that significantly improved the utility of powdered activated carbons for the separation and isolation of organic compounds (9). In these preparations, powdered carbon is dispersed on the surface of a finely divided material that is suitable for use in chromatography, thus reducing the difficulties of solvent flow and permitting the use of much smaller quantities of carbon. Supporting materials previously found to be most effective as dispersing matrices were, shredded polyurethane foam and a polystyrene/divinylbenzene copolymer bead (10) (Bio-Beads SX-3, Bio-Rad Laboratories, Richmond, CA). Other materials tested and found less suitable were sand, celite, Teflon beads, and glass beads. At the Columbia Laboratory, applications of activated carbon to compound and class separations have required the use of the adsorbent both in open (gravity flow) column chromatography and in pressurized multicolumn chromatography systems. The adsorbent of carbon dispersed on foam has been used to fractionate polychlorinated biphenyls (PCBs) into numerous structural classes and to isolate the minute quantities of highly toxic byproducts, polychlorodibenzofurans
(PCDFs), contained in these preparations (9, 11, 12). In pressurized chromatography systems, the utility of activated carbon in the separation of chemical classes and of compounds within a class has been significantly expanded. The dispersed carbon adsorbent has been incorporated in a sequential, multicolumn chromatography system (including columns containing a size exclusion gel and cesium silicate) that permits retention and fractionation of several important classes of environmental contaminants by adsorbents of widely varying but complementary selectivities (13). More recently, and reported herein, a significant improvement has been made in the preparation of dispersed carbon adsorbents by employing shredded glass fibers as the dispersing material, essentially eliminating a number of problems associated with other dispersing materials. EXPERIMENTAL SECTION Preparation of the Carbon/Glass Fiber Mixture and Packing of the Column. The desired amount of glass fiber filter material (Toyo GA200, Nuclepore Corp., Pleasanton, CA, or a similar product) is cut into small pieces (approximately 3 mm X 5 mm) with scissors and placed in methylene chloride, using approximately 50 mL for every 500 mg of glass fiber. The fibers in CHzClzare shredded for 15-30 s with a Polytron homogenizer (Brinkman Instruments, Westburg, NY). The desired amount of carbon is then added and the mixture thoroughly stirred until the carbon is uniformly distributed. The carbon is removed from suspension as it adheres to the settling glass fibers. In this laboratory, particulate carbon with a mean diameter of less than 20 hm has been used. Larger particles do not adhere to the fibers. In preparation of the chromatography column, a column is fitted with a glass funnel, and the slurry of carbon/glass in CHzClz is poured into the assembly. To hold the mixture in place in the column and ensure that no carbon particles move from the column, the mixture is bracketed at both ends of the column by three to five disks of glass fiber filters with a particle exclusion limit of less than 1 hm. The carbon/glass fiber mixture is packed in portions with a glass rod; usually 0.5-1 cm of adsorbent bed is packed at a time. The degree to which the adsorbent should be compressed during packing of the column depends on the backpressure desired during operation. RESULTS AND DISCUSSION The physical and chemical inertness of the glass matrix are essentially complete. In contrast to polyurethane foam, the glass fibers do not swell in solvents, are not a source of background interferences in the analysis of purified compounds, and are not subject to photolytic, oxidative, or other types of degradation. Background interferences attributed to the foam have occasionally been a significant impediment
This article not subject to US. Copyright. Published 1981 by the Amerlcan Chemical Society
