submitted papers

Emission Spectrometric Determination of Trace Metals in Biological Tissues*

John Webb, Wil l iam Niedermeier, and James H. Griggs

Division of Cl#tical Immunology and Rheumatology, University of Alabama School of Medicine, Birmingham, Alabama 35294 Thomas N. James

Cardiovascular Research and Training Center, University of Alabama School of Medicine, B#mingham, Alabama 35294 (l~eceived 19 Februa ry 1973)

An emission spectrometric method of analysis is described which permits duplicate analyses to be performed on 1-g specimens of tissue for 13 elements in the microgram percent range. Pig hearts were dissected into 17 separate pieces representing discrete anatomic sites. Samples were prepared for analysis by low temperature wet digestion. On the basis of their composition of the macro elements sodium, potassium, and phosphorus, these tissues could be divided into two groups: one including blood vessels and heart valves, and the other including ordinary and specialized myocardium. A method is described for adjusting the macro element composi- tion of these tissues to that of a common matrix, thus allowing trace metal analysis of both groups of tissues to be performed using only a single set of standards. A solution of the ash was placed in hollow cup graphite electrodes with center posts, evaporated to dryness, and analyzed in the dc arc with a Jarrell-Ash model 66,000 direct reading emission spectrometer. At concentrations above 10 ~g%, the coefficient of variation was about 10% for most of the trace metals studied. INDEX HEADINGS: Emission spectroscopy; Methods, analytical; Matrix effects; Trace metals; Biological material; Cardiovascular tissues.

I N T R O D U C T I O N

Recent studies from this labora tory have described methods for the determinat ion of t race metals in bio- logical fluids 1, 2 by direct reading emission spectroscopy. These methods have been used to analyze blood serum, synovial fluid, and saliva for 14 trace metals in concen- t ra t ions as low as 1 pg/100 ml. 3-5 The need to extend these studies to include other biological samples has become increasingly apparent . Obvious difficulties in analyzing samples of solid tissues include dissection wi thout introducing metal contaminants , and the necessity of minimizing or el iminating matr ix effects due to the var iable concentrat ions of macro elements in tissues of different origin.

Several recent reports have suggested t ha t cer tain t race metals may have a role in the etiology or patho- genesis of cardiovascular diseases. 6-~2 This communica- t ion describes an emission spectrometric method tha t is capable of analyzing cardiovascular tissues for concen- t ra t ions of t race metals as low as 1 pg/100 g of tissue.

* These studies were supported by Grant HL 11,310 and MIRU Contract PH 4367-1441 from the National Heart and Lung Institute and Grant AM 03555 from the National Institute of Arthritis and Metabolic Diseases and Grant ES 00836-01 from the National Environmental Health Sciences Institute.

I. EQUIPMENT AND MATERIALS

The operating parameters used with the Jarrell-Ash model 66,000 direct reading emission spectrometer are listed in Table I. A Simeon-Twyman 13 lens system was used to mask off emission from the incandescent elec- trode tips and illuminate the entrance slit of the spectrograph.

The 13 elements for which analyses were performed are listed in Table II along with the analytical lines used and the respective exit (secondary) slitwidths.

Standards were prepared from spectroscopically pure reagents which were purchased from Johnson-Matthey Company, London, England. Nitric, perchloric, and hydrochloric acids used for digestion, and ammonium chloride, which was used as a spectroscopic buffer, were special high pur i ty reagents purchased from E. Merck, A. G., Darms tad t , Germany. Dist i l led water was pre- pared from deionized water in a borosil icate glass distil- la t ion appara tus . All l abora tory glassware and the plastic and glass dissecting inst ruments were cleaned with reagent grade concentrated nitric acid mainta ined a t 100°C. This was followed by repeated rinsing in copious volumes of distilled water.

Pig hearts were obtained from a commercial slaugh- terhouse, refrigerated, and dissected within 3 h after

342 Volume 27, Number 5, 1973 APPLIED SPECTROSCOPY

TABLE I. Apparatus and spectrometric operating conditions.

Electrodes Sample ASTM P-1 Counter ASTM C-5 Analytical gap 4 mm Preburn 0 Exposure 50 sec

Source unit Jarrell-Ash Varisource continuous dc arc

Voltage 230V de Current 10A de

Spectrometer Jarrell-Ash 1.5 meter compact Atomcounter

Grating 30,0000 groves per inch Dispersion 5.6 A per nora1 Wavelength range 2000-8000 A Slitwidth

Entrance 25~ Exit 10~ or 75~

Readout DAC (Digital Automation Corp.) A-to-D converter and

interface with Western Electric Teletype and tape punch

TABLE II. Analytical lines used for elements studied.

Element Wavelength (A) Slitwidth (u)

Copper 3247.5 a 75 Aluminum 3961.5 75 Barium 5535.5 10 Manganese 4030.7 75 Nickel 3414.7 10 Cesium 4555.3 10 Tin 3262.3 75 Strontium 4607.3 75 Chromium 4254.3 75 Zinc 2138.5 10 Lead 2833.0 75 Molybdenum 3193.9 75 Cadmium 2288.0 75

,, Second order.

death. Any use of stainless steel instruments was shown in preliminary experiments to result in unacceptable contamination of the specimens. Therefore, all dissec- tions were done with borosilicate "knives" formed by shear cleavage of 1/~-in. glass rods. With practice, glass rods can be broken in such a way as to produce a sharp sliver tha t remains a t tached to the rod. "Blades" more than }~ in. long can be produced which are extremely sharp and satisfactory for the dissections required. Polypropylene (Nalgene) forceps were also useful in performing the dissections and were demonstrated to be free of any contaminating elements.

II. M E T H O D S

A. Sample Preparation

The heart with its appended blood vessels was dis- sected into the pieces representing the 17 sample sites listed in Table I I I . All surfaces exposed by the butcher ' s knife in the process of removing the hearts were care- fully avoided. The tissues were placed into preweighed ignition tubes (20 X 150 ram), sealed with plastic film (Saran Wrap) to prevent water loss by evaporation, and prompt ly weighed to an accuracy of 0.1 mg. Sample size

varied from several grams for the free ventricular wall to about 250 mg for the heart valves. High pur i ty digestion acids (1 ml of nitric acid and 0.25 ml of per- chloric acid) were added to each tube. The vigorous reaction between the tissues and acids at even slightly elevated temperatures required tha t the digestion proceed at ambient t empera ture for about 24 hours before the tubes were placed in heating blocks and slowly heated to and maintained at a tempera ture of 130°C for about 24 hours. Several additions of the nitric and perchlorie acids followed by additional periods of heating often were required to complete the diges- tion.

When digestion of the specimens was complete, the colorless solutions were heated a t 180°C until dry (5 to 6 h). To convert the metals to their chlorides, 1 ml of high pur i ty hydrochloric acid was added to each of the cooled samples and the contents of the tubes were again evaporated at 130°C until dry. The ash was re- consti tuted to a volume equal (in ml) to one-quarter of the mass (m, in g) of the wet tissue with a solution containing spectroscopic buffer (1.8 % NH4C1) and the matrix components discussed below. A fourfold con- centration of the metals was thus achieved. Subse- quently 0.1 ml of the sample was transferred to the crater of the sample electrode with an acid-washed disposable micropipet (Microcaps purchased f rom Kensington Scientific Corporation) and evaporated to dryness at reduced pressure in a vacuum desiccator a t room temperature. ~ h e n 1 g or more of tissue was available, the specimens were analyzed in duplicate. Results were expressed as the mean of the two determi- nations. The ash of tissues whose mass was less than 400 mg could not be reconsti tuted by the above procedure since m/4 < 0.1 ml. In such cases the ash was reconsti- tuted to 0.1 ml, a single analysis was performed, and the data were corrected for the additional dilution.

B. Preparation of Spectroscopic Buffer-Matrix Solution

Five pig hearts were dissected into the 17 pieces shown in Table I I I and ashed by the method described above ("Sample Prepara t ion") . Sodium and potassium were determined by flame emission spectroscop~ using a flame photometer (Eppendorf, model 700, Hamburg , Germany) . Phosphorus was determined by the method of Fiske and SubbaRow. 14 As shown in Table I I I , the tissues of the 17 sample sites can be divided into two groups on the basis of their content of these three macroelements. The blood vessels and heart valves (group I) are characterized by high sodium, low potas- sium, and low phosphorus values. The remaining tis- sues which are composed essentially of ordinary and specialized myocardium (group I I ) are characterized by ]ow sodium, high potassium, and high phosphorus content. The AV (atrioventricular) node and His bundle, however, had a relatively low potassium con- tent. The mean concentrations, respectively, for groups I and I I were 7.2 and 3.9 mmoles of sodium, 3.8 and 5.3 mmoles of potassium, and 2.5 and 5.1 mmoles of phos- phorus per 100 g of tissue. The differences in composi- t ion between these two groups of tissues were evaluated statistically by the nonparametr ic Mann-Whi tney U-

APPLIED SPECTROSCOPY 343

TABLE III. Matrix composition of cardiovascular tissues. •

Sodium Potassium Phosphorus

Mean Range S.D. Mean Range S.D. Mean Range S.D.

Group I Right superior vena cava 5.1 2.3-6.8 1.8 3.0 1.0-4.7 1.6 Aorta 6.0 5.0-6.5 0.6 2.7 2.1-3.2 0.5 Main pulmonary artery 6.4 5.5-7.0 0.6 3.2 2.2-4.2 0.7 Mitral valve 8.9 7.6-12.4 2.1 3.2 b 1.9-4.1 1.0 Tricuspid valve 7.8 6.3-8.8 0.9 4.1 3.0-4.6 0.6 Aortic valve 8.5 6.8-10.0 1.4 3.4 2.6-4.6 0.8 Pulmonary valve 7.8 6.2-9.1 1.2 3.6 2.4-5.2 1.2

Group II Right atrium 4.5 3.5-5.8 0.8 4.7 2.2-6.1 1.5 Left atrial appendage 4.7 3.9-5.4 0.6 5.3 4.0-7.3 1.4 Right ventricle (free wall) 4.2 3.4-5.2 0.7 5.9 5.0-7.0 1.3 Left ventricle (free wall) 3.4 2.9-3.8 0.5 5.9 3.6-7.4 1.6 Sinus node 4.3 1.9-5.5 1.4 4.2 1.9-6.6 2.2 Atrioventricular node and His bundle 4.1 2.8-4.9 0.9 2.9 0.5-5.4 2.1 Left ventricle--papillary muscle 3.4 3.1-3.7 0.2 6.7 4.1-8.1 1.6 Crista supraventricularis 4.4 3.5-4.9 0.5 6.1 5.6-6.7 0.4 Interventricular septum 3.4 3.3-3.5 0.1 6.8 4.7-7.6 1.2 Left bundle branch 3.9 3.7-4.1 0.2 6.3 5.7-7.0 0.6

3.0 0.3-5.6 2.4 2.1 1.1-2.9 0.7 2.2 1.0-2.8 0.8 2.5 2.0-2.8 0.3 2.2 1.6-2.9 0.6 2.2 1.0-3.0 0.8 2.8 1.3---4.2 1.3

4.7 3.6-5.3 0.7 4.8 4.2-5.4 0.5 5.3 4.4.--7.7 1.5 5.5 3.0--8.4 2.0 3.9 1.3-5.2 1.6 4.0 3.0-4.9 0.9 6.3 5.0-7.3 0.9 5.8 3.7-7.1 1.4 6.0 2.5-7.5 2.0 6.2 5.8-6.5 0.4

Concentrations are expressed as mmoles of metal per 100 g of tissue (wet weight). b Four determinations only.

test and the Student's t-test. By both tests the differ- ences in phosphorus and sodium content between the two groups were significant at the 0.I % probability level. Differences in potassium were significant at the 0.i % level by the t-test and the 1% level by the non- parametric test. Variations of this magnitude in matrix composition could result in significant errors in emission spectrometric analyses. Consequently, the macroele- ment compositions of the buffer solutions used to dis- solve the ash of the two groups of tissues were adjusted to compensate for these differences. Thus, following reconstitution, the ash from both groups of tissues had the same macro element composition as the standard solutions described below, which contained 564.8 mmoles of Na, 212.4 mmoles of K, and 204.8 mmoles of P per liter. These values reflect the fourfold increase in concentration effected by the ashing technique. The matrix solution used to reconstitute the ash from group I tissues contained 276.8 mmoles of Ha, 60.4 mmoles of I{, and 103.2 mmoles of P per liter. The matrix solu- tion for group II tissue contained 408.8 mmoles of Na per liter. Both of these matrix solutions also contained 1.8 % NH4CI, which served as a spectroscopic buffer. A volume equal to m/4 ml of the appropriate matrix solu- tion when added to the tissue ash resulted in a matrix with composition equal to that of the matrix in which the standards were prepared. This matrix composition will be referred to as the standard cardiac matrix.

C. Preparation of Standards

The matr ix solution in which the s tandards were prepared contained 4 t imes more sodium than normal blood serum, and 4 times more potass ium and phos- phorus than were found in group I tissues in order to account for the fourfold increase in concentrat ion of the samples achieved during the ashing procedure. Spectroscopically pure NaC1, KC1, and KH2P04 were used.

The concentrations of t race elements are expressed in terms of ug/100 g of s tandard , commonly called microgram percent (pg%). One microgram percent is the equivalent of 0.01 ppm. The s tandard solutions of the trace elements were prepared by dilut ing a 0.02 % stock solution of the elements to a concentrat ion of 1000, 400, 200, 40, 20, and 4 vg % with matr ix solution. Thus, 0.1 ml of each of the six s tandard solutions was equivalent to 0.1 ml of tissue ash solution derived from 0.4 g of tissue tha t contained 250, 100, 50, 10, 5, and 1 ug of the trace element/100 g. Each of these s tandard solutions contained all 13 of the t race elements.

In order to minimize errors due to possible minute impuri t ies present in the reagents used for ashing, the s tandards were t rea ted by the procedure described for the tissue specimens. The dried residue of metal chlo- rides was dissolved in a 1.8% NH4Ci spectroscopic buffer solution and made to the original volume of un- ashed s tandard. Electrodes were prepared b y trans- ferring 0.1 ml of the ashed s tandard solutions (equiva- lent to 0.4 g of tissue) into the crater of the sample electrodes and evaporat ing to dryness in a vacuum desiccator.

D. Spectrometric Procedure

The ins t rument was cal ibrated each day on the basis of quadrupl icate determinat ions performed on each of the 6 s t andard solutions. The equations of the curves t ha t described the concentrat ion response of the instru- ment for each of the elements were calculated with the use of a Sigma 7 computer system (Scientific D a t a Systems). Using these cal ibrat ion curves derived from the standards, the computer then calculated the con- centrat ions of the elements in the unknown specimens? Concentrat ions were expressed in terms of micrograms of the element per 100 g of sample (ug%).

344 Volume 27, Number 5, 1973

I I I . R E S U L T S A N D D I S C U S S I O N

The distribution of the matr ix elements Na, K, and P among the 17 anatomical ly discrete sample sites in these hearts is presented in Fig. 1. Classification of the tissues into two groups on the basis of their content of these three elements reflects apparent functional differ- ences. Group I tissues, which contain high sodium, low potassium, and low phosphorus concentrations, repre- sent blood vessels and heart valves which are rich in connective tissues and extracellular fluid. Group I I tissues, which contain low sodium, high potassium, and high phosphorus concentrations, represent ordinary and specialized myocardium, tissues rich in cells and comparat ively low in extracellular components.

The effect of differences in matrix composition on spectrometer response is i l lustrated in Fig. 2. These curves represent the ins t rument response to nickel in the presence of the standard cardiac matrix and in matrices containing sodium, potassium, and phos- phorus in the mean concentrations of group I and group II tissues. These curves indicate that, as with the other elements, serious errors could be introduced into the analysis if the matrix effects were ignored.

Typical calibration curves for copper, nickel, and lead in the standard cardiac matrix are shown in Fig. 3. As these curves illustrate, some elements could be de- termined at lower concentrations than others. In view of the procedure used to establish the composition of the cardiac matrix, it is not surprising that the spec- trometer response is in close agreement with that reported earlier for standards prepared in blood serum matrix.2

Previous studies 2 have shown that better analytical responses were obtained for many trace metals in the presence rather than in the absence of sodium chloride. Consequently, sodium chloride was added to the stand- ards and the ashed tissues to increase their concentra-

2

o N HN 4

2

H tHH o M 4

2

o 2

FIO. 1. Distribution of

NO

I1H 1,11 6 8

K

H H HHH M 6

P

M HHHNN N 4 mmoles/, oo6g

sodium (Na), potassium (K), and phosphorus (P) levels (mmoles/100 g) in 17 different cardio- vascular tissues. The hatched bars indicate the data for the three blood vessels and four heart valves. The open bars indi- cate the data for the I0 other tissues.

I000

COUNTS

I00

,ol I

I I I

IO ioo 250 ~g%

FIG. 2. Calibration curve for nickel (Ni) over the range 1 to 250 ~g% in the standard cardiac matrix (curve a), the matrix of group I I tissues (curve b), and the matrix of group I tissues (curve c).

500C

IOOC

COUNTS

IOC

Cu

Ni

Pb

I I I

I0 I00 250 iJg%

FIG. 3. Calibration curves for copper (Cu), nickel (Ni), and lead (Pb) over the range 1 to 250 ~g% in the standard cardiac matrix.

tions of sodium to levels shown previously in studies on blood serum S to result in acceptable detection limits. Potassium and phosphate were added to the standards and to group I tissues to increase their concentration of these elements to tha t of group I I tissues. This approach resulted in improved spectrometric response and allowed analyses to be performed on two groups of tissues with divergent matrix compositions using a single set of standards.

The use of emission spectroscopy for the analysis of biological materials has been reported by a number of investigators. 16-24 In one of the most extensive stud- ies 19-21 in which this method has been applied, 29 differ- ent human tissues from 150 adult subjects were analyzed for 19 trace metals and 5 macro elements. These studies have generally been done using a single matrix for all tissues. As shown in the present report, this procedure could lead to serious errors.

In a s tudy to determine the precision of the method, nine determinations were made on each of the six stand-

APPLIED SPECTROSCOPY 345

ard solutions. After the usual computer computations on the standards, these data were treated as unknowns. The standard deviations of the computed concentra- tions of these nine samples were then calculated. They are summarmed in Table IV. Standard deviations of all elements were higher for the more concentrated solu- tions, but, when calculated on the basis of the percent of the concentrations of the element in the standard (coefficient of variation), the best results were obtained at concentrations of 50 or 100 ~g %. At concentrations of 50 pg% or greater the standard deviations were generally less than 10 % of the concentration.

The results of analyses of normal pig aorta and myo- cardium taken from the free wall of the ]eft ventricle (11 hearts) are shown in Table V. The number of specimens (N) containing a particular element is listed together with the mean and the range of the values observed. As in the case of the serum trace metal data previously reported, 2 the mean values were calculated assuming a zero value for those determinations in which the concentration was below the limit of detection. For most of the elements the mean concentrations in both of these tissues is at least two times greater than the respective threshold value? Inspection of the sum- marized data in Table V indicates that the procedure

TABLE IV. Standard deviations obtained by emiss ion spec- trometric analys is of trace meta l s in standard cardiac matrix.

Element Solution concentration (,g%)

1 5 10 50 100 250%

Copper Aluminum Barium Manganese Nickel Cesium Tin Strontium Chromium Zinc Lead Molybdenum Cadmium

0.23

i:i 0.8

0.4 0.7 2.2 7.0 12.5 4.4 4.0 15.8 44.4

2:8 2.6 2.6 7.1 42.9 1.2 1.3 3.4 6.2 16.7 2.4 3.6 7.9 11.9 41.2 3.4 3.2 4.1 10.2 24.9 1.7 1.8 2.0 7.2 12.9

4.3 3.5 7.1 45.5 (}:9 2.4 8.4 9.5 12.6

... 18.4 31.7 74.3 i l i 1.6 4.1 10.8 59.1 2.9 2.1 6.3 6.3 24.8 1.8 1.5 3.2 7.9 93.6

TABLE V. Trace meta l concentrat ions in porcine aorta and myocardium.

Aorta Myocardium

Na Range Mean N Range Mean (P*g %) (Dg %) (.~g %) (ug %)

Copper 11 25-55 40 n 119-280 182 A luminum 8 < '-170 57 6 <6-200 40 Bar ium 51 4-20 51 5 <3-5 <3 Manganese 11 2-17 7 11 4-15 8 Nickel 8 <1-5 2 10 <1-5 3 Cesium 0 <4-57' 5 1 <4-6 <4 Tin 11 7-27 16 11 29-65 40 Stront ium 11 3-52 6 9 < .5-7 2 Chromium 8 <1-8 3 5 <1-8 2 Zinc 51 40-080 500 11 70-1000 504 Lead 8 <2-42 16 50 <2-30 11 Molybdenum 5 <2-4 <2 9 , <2-11 5 Cadmium 50 <2-36 10 11 3-32 10

a At: number of specimens containing the element.

3 4 6 Volume 27, Number 5, 1973

described in this paper is capable of determining these trace metals at the concentrations in which they appear in biological tissues. A detailed account of the analyses for the 13 trace metals in the entire series of 17 different cardiovascular tissues (Table I I I ) will be reported else- where. 15

Although this study was limited to tissues of the heart and blood vessels, it appears to be generally applicable to tissues from other sources. Most connec- tive tissues and muscle probably have matrices com- parable to tissues of groups I and II , respectively. 2° The matrices of other specialized tissues are probably quite different, however, and must be determined before accurate quantitative spectroscopy can be achieved. Preliminary studies on calcified tissues such as bone and teeth indicate that they present special problems. The extremely high concentrations of calcium and/or phosphate appear to suppress emission from the trace metals. Studies on these tissues would probably require extraction procedures which are extremely difficult to accomplish without contamination of the specimen with extraneous metals.

The use of stainless steel dissecting instruments re- sults in serious contamination of the specimens. The borosilieate glass and plastic instruments used in this study have limited capabilities and are suitable only for postmortem dissections. We are presently investigating the use of surgical instruments composed of highly refined titanium, thus malting it feasible to obtain uncontaminated samples of both normal and patho- logical human tissues removed in the course of surgical procedures.

A C K N O W L E D G M E N T

The authors wish to thank Patrick Campbell for his assistance in the sodium, potassium, and phosphorus analyses.

1. R. S. Johnson, W. Niedermeier, J. H. Griggs, and J. F. Lewis, Appl. Spectrosc. 22,552 (1968).

2. W. Niedermeier, J. H. Griggs, and R. S. Johnson, Appl. Spectrosc. 25, 53 (1971).

3. W. Niedermeier and J. H. Griggs, J. Chronic Dis. 23,527 (1971).

4. W. Niedermeier, W. Prillaman, and J. H. Griggs, Arthritis l~heum. 14, 533 (1971).

5. S. Dreizen, B. M. Levy, W. Niedermeier, and J. H. Griggs, Arch. Oral. Biol. 15,179 (1970).

6. F. W. Sunderman, Jr., S. Nomoto, A. M. Pradhan, It. Levine, S. H. Bernstein, and R. Hirsch, New Engl. J. Med. 283,896 (1970).

7. It. A. Schroeder, A. P. Nason, and I. II. Tipton, J. Chronic Dis. 23, 123 (1970).

8. M. Slavin, Emission Spectrochemical Analysis (Wiley- Interscience, New York, 1971).

9. H. A. Schroeder, J. Chronic Dis. 12,586 (1960). 10. M. D. Crawford, M. J. Gardner, and J. N. Morris, Lancet

1,827 (1968). 11. R. Masironi, Bull. W.H.O. 40,305 (1969). 12. Anon., W.H.O. Chronicle 26, 51 (1972). 13. F. Twyman and F. Simeon, Trans. Opt. Soc. (London)

31,169 (1930). 14. C. H. Fiske and Y. SubbaRow, J. Biol. Chem. 66, 375

(1925). 15. J. Webb, W. Niedermeier, J. It. Griggs, T. N. James, K.

Kirk, and M. Turner, submitted for publication.

16. R. Monacelli, H. Tanaka, and J. II. Yoe, Clin. Chim. Acta 1,577 (1956).

17. L. M. Paixao and J. If. Yoe, Clin. Chim. Acta 4,507 (1959). 18. R. E. Nusbaum, E. M. Butt, T. C. Gilmour, and S. L.

Didio, Am. J. Clin. Pathol. 35, 44 (1961). 19. I. H. Tipton, M. J. Cook, R. L. Steiner, C. A. Boye, If. M.

Perry, Jr., and H. A. Schroeder, Health Phys. 9, 89 (1963). 20. I. H. Tipton and M. J. Cook, Health Phys. 9,103 (1963).

21. I. H. Tipton, It. A. Schroeder, H. M. Perry, Jr., and M. J. Cook, Health Phys. 11,403 (1965).

22. E. M. Butt, R. E. Nusbaum, T. C. Gilmour, and S. L. Didio, Arch. Environ. Health 8, 52 (1964).

23. H. R. Imbus, J. Cholak, L. H. Miller, and T. Sterling, Arch. Environ. IIealth 6,286 (1963).

24. A. J. Bedrosian, R. K. Skogerboe, and G. H. Morrison, Anal. Chem. 40,854 (1968).

Application of Electron Emission Spectroscopy to Charaeterize Sulfur Bonds in Coal

Harry D. Schultz

United States Department of the Interior, Bureau of Mines, Morgantown Energy Research Center, Morgantown, West Virginia 26505

W. G. Proctor

Varian Associates, 611 Hansen Way, Palo Alto, California 9~303

(Received 28 January 1971 ; revision received 22 March 1973)

Electron binding energies of minerals typically found in coal were measured by the induced electron emission method. The shift in binding energies for Fe and S atoms were correlated with different chemical environments. The iron 2p3/~ electron in pyrrhotite (Fex_.S) is bound by 3.8 eV more than the Fe 2p3n electron in pyrite (FeS2), whereas the 2p sulfur electron is shifted by 0.8 eV. Chemical shift data for minerals and organic sulfides were determined and correlated with structure variations. Subsequently, multiple sulfur signals observed on raw coals were correlated with this IEE data. Thus, we have demonstrated that the modes of occurrence of sulfur (pyritic, sulfate, organic) ct~n be determined on raw coal; this represents the first spectroscopic technique capable of performing this characterization. INDEX HEADINGS: Electron emission spectroscopy; Sulfur; Coal.

INTRODUCTION

Electron emission spectroscopy is a method for determining the absolute vMues of the binding energies of electrons in atoms. The measured values are de- pendent on the chemical environment of the specific a tom under study; that is, ~ chemical shift occurs. In a molecule the chemical shift for a specific element de- pends on the electron binding energy of the atomic shell under investigation and type of bonding partner. Con- sequently, the shifts are characteristic not only of the specific elements but also of the functional group and structural composition of the molecule. Electron bind- ing energy shifts increase with increasing electro- negativity difference between the atoms in ~ bond. Chemical shifts have been measured in many of the light elements--nitrogen, sulfur, oxygen--and correla- tions established between the binding energy and the charge on the atom as a function of valence state, co- ordination number, etc. 1 Chemical shifts as high as 10 eV have been reported for the same atom in different chemical environments.

The electron emission spectra are characterized by specific signals occurring at energies that correspond to

atomic energy levels. The kinetic energy of a photo- electron, Ek, is related to the orbital binding energy, Eb, of the ejected electron by

Ek = h~ -- Eb (1)

where hv is the energy of the photons used for irradiating the sample. Much of the theory of photoelectron spectroscopy-is still primitive compared to other spec- troscopic techniques. However, the potential of photo- electron spectroscopy for chemical characterization is great.

In this work, correlations of electron binding energies with environmental structures are reported for sulfur compounds typically found in coal. The use of electron spectroscopy to distinguish various bonding types of sulfur in raw coal is presented for the first time. In addition, the possibility of using electron spectroscopy to investigate crystalline structure differences in minerals is discussed.

I. EXPERIMENTAL

The electron spectrometer used in our measurements is a Varian Associates' V- IEE 15 Induced Electron

Volume 27, Number 5, 1973 APPLIED SPECTROSCOPY 347