cantly even by boiling concentrated sulfuric acid but is easily charred thermally on heating to dryness. The resulting char can then be oxidized completely and safely with a mixture of nitric and perchloric acids. Sodium acid sulfate is added as an acid buffer to prevent hydrolysis and thermal decomposition of lead and bismuth sulfates during heating to dryness and to prevent precipitation of very insoluble anhydrous sulfates of iron and other metals that might be extracted. Reduction and Volatilization of Lead. While checking the applicability of the potassium fluoridepyrosulfate fusion in platinum (10) to the direct decomposition of solid samples prior to the determination of lead, several per cent of the lead-212 tracer being used was always unaccounted for, presumably lost by volatilization. I n addition, the platinum dish itself always contained several per cent of the tracer after the fluoride fusion, only part of which could be removed in the subsequent pyrosulfate fusion. The remainder could not be removed by any
means. Apparently, some of the lead is reduced to the metal in the slightly alkaline fluoride fusion by organic matter in the sample or by reducing gases in the burner used to make the fusion, some of which is then volatilized a t the high temperature employed and some is permanently alloyed with the platinum dish. This residual 10.6-hour lead-212 tracer decays off in a few days and the dish can be put back into service but it is obvious that any contamination of expensive platinum dishes by 22year lead-210 is intolerable. Therefore, samples should be wet-ashed vigorously in glassware to oxidize all organic matter completely and to leach out as much of the lead activity as possible before fusing the residue in platinum, particularly when relatively radioactive samples such as ores, mill tailings, etc., are being analyzed. The preliminary acid treatment has the additional distinct advantage of leaching out other easily reducible oxides and phosphates, both of which are particularly corrosive to platinum ware.
LITERATURE CITED
(1) Adams, J. A. S., .Lewder, W. M.,
“The Natural Radiation Environment,” University of Chicago Press, Chicago,
1964. (2) Analytical Methods Committee, Analyst 84, 127 (1959). (3) Aronson, A. L., Hammond, P. B., Nucleonics 22, No. 2, 90 (1964). (4) Bambach, K., Burkey, R. E., IND. ENG.Cnm.. ANAL.ED. 14. 904 (1942).
(5) Code of Federal Regulations,‘ Titie 10, Part 20, Federal Register, Nov. 17, 1win
(6)-Gibson, W. M., “The Radiochemistry of Lead,” National Academy of Sciences, Nuclear Science Series, NAS-NS 3040 (1961). (7) Holtzman, R. B., Health Phys. 9, 385 (19fIR\. \----,.
(8) Jaworowski, Z., Nukleonika 8, No. 5, 333 (1963). (9) Maynes, A. D., McBryde, W. A. E., ANAL.CHEM.29, 1259 (1957). (10) Sill, C. W., Ibid., 33, 1684 (1961). (11) Sill, C. W., Willis, C. P., Ibid., 36, 622 (1964). (12) Ibid., 37, 1176 (1965). (13) Sill, C. W., Willis, C. P., Flygare, J. K., Jr., Ibid., 33, 1671 (1961). (14) Talvitie, N. A., Garcia, W. J., Ibid., 37, 851 (1965). RECEIVEDfor review July 19, 1965. Accepted October 7, 1965.
An X-Ray Fluorescence Method for the Determi nutoi n of Ca Icium, Potassiu m, ChI ori ne, Sulfur, and Phosphorus in Biological Tissues GEORGE V. ALEXANDER laboratory of Nuclear Medicine and Radiation Biology, University o f California, 10s Angeles, Calif.
b An x-ray fluorescence method for the determination of calcium, potassium, chlorine, sulfur, and phosphorus in dried biological tissues, fluids, and organic extracts from tissues has been developed. The procedure has been developed and substantiated by the use of synthetic standards representing the extremes of concentration found in biological systems. Beyond the determination of a sensitivity constant for each element the method requires only the application of an absorption correction. This correction is determined in part by measuring the attenuation of Ti Ka x-rays by the sample and in part by the determined concentrations. Details of the method, including examples, are presented.
T
GENERAL LACK of suitable procedures for determining sulfur in a broad range of organic-based materials such as is found in biological tissues, fluids, and extracts prompted HE
the development of this x-ray fluorescence procedure. A number of x-ray procedures have been developed for sulfur but in each case have been applied to such a limited range of samples-e.g., gasoline ( 5 ) , oil (4), oil additives (a), or blood serum (7)-that the possible extension to other organic materials without resorting to an infinitely complicated set of standards has been doubtful. Sulfur, chlorine, and potassium are lost totally or in part during the thermal ashing of biological tissues at temperatures above 400’ C. This is also true for sulfur when tissues are ashed by low temperature-activated oxygen techniques. To avoid these losses it is necessary to carry out the analysis on fresh or dry tissue. The purpose of this paper is to present the details of an x-ray fluorescence procedure suitable for determining sulfur, calcium, potassium, chlorine, and phosphorus in biological tissues and organic components separated from these tissues.
90024 EXPERIMENTAL
Apparatus. The x-ray fluorescence equipment used for this work is commercially available from the North American Phillips Co. T h e x-ray tube is a tungsten target FA-60 which is mounted to irradiate the sample from above. The lead aperture in t h e conventional tube was replaced with a small trapezoidal mask which limits the irradiated area a t the sample surface to 0.5 X 0.5 inch. The x-ray supply voltage and current are regulated to within *0.25% and *0.10%, respectively. The spectrometer is equipped with a 2-inch, 0.125-inch spacing entrance soller slit, a flat ethylenediamine dextrotartrate (EDDT) crystal (2d = 8.808A.), a 4-inch1 0.007-inch spacing exit soller, and a P-10 gas flow proportional counter. The standard entrance soller slit is modified with apertures so that only the 0.5- X 0.5-inch sample area can be viewed by the spectrometer. The entire system is enclosed in a helium atmosphere mainVOL. 37, NO. 13, DECEMBER 1965
* 1671
Table I.
Typical Background Conditions
Position Ca KCY K KCY B,*
Wavelength, A. 3.360 3.744 4.030 4.729 C1 K a 5.373 s Ka 2.750 Ti Ka (2) 6.155 P Ka e Assuming that i V ~ i= 600 C.P.S. and * Detd. by interpolation.
fA 1.42 1.11
1.00 0.549 0.444 0.436b 0.407 E,* = 1.83 c.p.s.
tained by the continuous flow of helium a t a rate of approximately 5 cubic feet/ hour. The sample planchet is a cavity etched into the surface of a titanium sheet. The construction of this type of planchet required the solution of two problems: masking the planchet against the hydrofluoric acid etchant and determining an acid concentration which would minimize the tendency to undercut the masked surface. After attempting to mask the planchet with various types of tape with only occasional success we obtained a small quantity of Turcoform hfask No. 505 (Chem-Mill, Wilmington, Calif.) which, when thinned with xylene and applied in two air-dried coats, defined the area to be etched very satisfactorily. High concentrations of hydrofluoric acid will undercut the mask a t a rate greater than the rate of penetration into the metal and results in sloping sides to the cavity. A mixture of concentrated hydrofluoric and nitric acids at a level of 25% and 15% by volume, respectively, will etch titanium a t a satisfactory rate while maintaining the rate of undercut a t a minimum. The surface of the shaped planchet was smoothed with 120-grit silicon carbide paper. The mask coats were applied and allowed to dry. The masked planchets were placed in the etching solution a t 70" to 80" C. with the surface to be etched facing upward for 20 seconds and then placed in distilled water. The cavities prepared in this manner were from 3.4 to 4.0 mils in depth. The signal is measured without pulse height discrimination by a conventional scaling circuit capable of handling count rates of 10,000 c.p.s. without serious coincidence loss. To maintain stable operation of all components, it was necessary to maintain the room a t 76" =k 6" F. Method. The tissue or fluid to be analyzed is lyophilized as soon after collection as is possible. After drying, the tissue samples are ground with a plastic pestle and a plastic vial by shaking in a Mixer-Mill (Spex Industries, Metuchen, N. J.) for 1 to 2 minutes. An aliquot from the tissue powder or lyophilized fluid weighing approximately 25 mg. is transferred to a 2-ml. sapphire grinding vial and ground by shaking with a sapphire ball for 2 minutes in a Wig-L-Bug (S. S. White Dental Mfg. Co.) amalgamator. The finely ground powder is weighed 1672
ANALYTICAL CHEMISTRY
fTi 0.00114 0 0
0.00043 0.00129 0
0.00048 BR = 0.50 C.P.S.
Bs
+ BT~O
C.P.S. 3.27 2.08 1.83 1.26 1.59 0.80 1.03
into the 0.5- X 0.5- X 0.0037-sample cavity of the "pure" titanium planchet. The powder is distributed evenly over the area and the surface is then smoothed with a small paddle made of Teflon. The amount of sample used depends largely upon the physical characteristics of the powder, but is generally in the range of from 10 to 20 mg. Smaller samples require more than routine patience. The loaded planchet is placed in the spectrometer and the system is allowed to zquilibrate for 1 to 2 minutes prior to measurement. The fluorescent radiation a t Ca KCY,K K a , 4.03 A., C1 K a , S K a , Ti KCY(n = 2) and P K a is measured in that order.
Other background values show a decline in count rate as the sample area is covered with organic materials. A linear decline of these backgrounds is observed when compared with the attenuation of the Ti K a (n = 2) signal observed through the sample i.e., BTi = fTi
*
NTi
(2)
where NTi is the net count for Ti K a (n = 2) after subtracting BR. The scatter backgrounds for these positions are determined by extrapolating the values to NTi = 0. The total background a t any point is then
A typical set of background values is shown in Table I. The titanium scatter factors, fT,, are largest in the regions nearest to the Ti signals, suggesting the need for a high resolution system for this analysis. Absorbance. The principal reason for using a titanium planchet is to have available a relatively soft x-ray, Ti K a (2.750 A ) ,with which to estimate the absorption by the sample of the soft x-rays from Ca, K, C1, S, and P. The absorbance is determined according to the equation :
BACKGROUND
The unwanted signal or background arises from three sources: the normal counter background, BE; the scatter background, B,, produced by scattering the relatively hard tungsten x-rays from the planchet and sample surfaces; and the background from the soft Ti K a and Kp x-rays, B T ~which , are not completely resolved by the monochromator system from the signals due to the elements of interest. The accuracy with which these backgrounds can be determined automatically determines the limit of sensitivity of the method. The value of BRis relatively constant and is kept a t a minimum by using as low a counter voltage as is practicale.g., 80 volts above the knee of the plateau for the longest wavelength to be detected, P Ka. With our detector this requires a voltage of 1675 volts. At several points within the wavelength range covered for the ayalysis there is little change in the background as the cavity is filled with organic materials such as glycine, stearic acid, starch, etc. The background a t 4.03 A. behaves in this manner and apparently represents in its entirety scattering of the relatively hard tungsten radiation by the planchet and to some degree the sample surface. All values of scatter background, B,, are referenced to this value according t o the equation where B,* is the scatter background a t 4.03 A. and f A is a constant.
where NT,O is the Ti K a signal from a bare planchet, NT,is the titanium signal with the sample in place and F T i is the titanium signal arising from planchet surfaces beyond the edge of the sample cavity. This is determined by blocking the signal from the cavity with lead. The value of F T , is equal to a constant-e.g., 0.0214-times the value of ~VT?. A group of 50 standards was prepared from calcium carbonate, potassium phthalimide, o-chlorobenzoic acid, cystine, Dbo-phosphoserine and glycine. The elements of interest were varied singly or in binary mixture. The only correction needed to convert the net count rates, N , as modified by the absorbance measurement, AT,, to concentration was a correction for differential absorption. This correction is necessary only when an absorption edge occurs between Ti K a and the wavelength being measured. This situation was verified when the data were found to agree with the equation:
where C is the element concentration, TY is the sample weight, N is the net signal from the element being determined, k is a constant, /J and PT, are the calculated mass absorption coef-
ficients (6) for the standards a t the wavelength being measured and a t T i K a . Figure 1 shows this relationship as observed for phosphorus when in binary mixture with various quantities of calcium, potassium, chlorine, sulfur, and sodium-containing compounds and with glycine. The p / p T , ratio for glycine is identical to that calculated for tripalmitin, sucrose, and other biologically possible combinations of C, H, E,and 0. The value of yo a t this point is taken as the sensitivity constant, y t . The correction for the presence of an absorption edge interposed between Ti K a and the wavelength being measured can be calculated from absorption coefficient tables. Equation 6 expresses the relationship between the concentration of interfering element and its effect upon the absorption ratios. ‘pt =
PO
k k
. G/PT,) Glycine WPTJSample 1
+
C (6)
where C is the concentration of interfering element and y is the calculated constant relating the concentration or concentrations to the change in absorption coefficient ratios. Values of this constant as derived from mass absorption coefficient tables (6) are shown in Table 11. With these constants it is possible to relate the true concentration to the observed quantities as follows:
(7) where p = 1
+ By
C
This equation is solved for each element by iteration to arrive at the true concentration. Generally no more than three cycles are required to arrive a t a value within 1% of thcl true value. It is most expedient to start with the calcium signal and procede to the phosphorus signal while adjusting the value of p according to the estimated concentrations resulting from the previous cycle. After determining the constant k and consequently p t for each element all standards were analyzed for Ca, K, C1, S, and P. These standards included binary mixtures of the elements as well as mixtures composed of all five elements in a glycine matrix. The concentration range covered was Ca: 0.02 to 0.70%, K : 0.3 to 3%, C1: 0.4 to 5%, S: 0.3 to 8% and P : 0.2 to 6%. The lack of effect of “neutral” diluents was verified by diluting a nominal standard (0.3% Ca, 1.5yo K, 27; C1, 1.5% S, and 1% P) with 20% by weight glycine, lithium carbonate, sodium carbonate, and barium carbonate. The correspondence between the theoretical values and the observed values together with the relative standard deviation for the ratio are
Table II.
Absorbance Interference Constant, y,
(%)-l
Interfering Element Wavelength
P Ka S Ka
C1 K a K Ka Ca KCY
P
S
c1
K
Ca
0.086
...
0.104 0.104
... ...
...
0.117 0.117 0.116
...
...
0.153 0.153 0.150 0.148
0.169 0.168 0.162 0.158 0.155
...
...
...
tabulated in the second column of Table 111. The mean square relative standard deviation for a single analysis is shown in the third column.
Table 111.
Element Ca
DISCUSSION
The apparent adequacy of Equation 7 seems to eliminate concern for any significant effect resulting from interelement secondary fluorescence. The levels of error indicated in Table I11 are well within those expected to result from variations in standards preparation, planchet loading, weighing, and general counting statistics. The effect of sample thickness was tested with special planchets having cavity depths of from 0.7 to 118 mils, The sample thickness was limited only by the accuracy with which A T , can be measured. I n general this requires that the weight be kept below 38 mg. (24 mg./cm.2). The absorbance per milligram, AT,/JY, ranged from 0.025 to 0.047 for biological tissues and organic compounds separated from these tissues. The range covered by the standards was from 0.025 to 0.068. The grinding requirements for the sample are two-fold. The sample powder must be fine enough to permit a
K
c1
S
P
...
Experimental Accuracy and Precision-Standards
yo Theoretical % Observed 1.012 1.043 1,000 0.964 0 990
f ,046 f ,006
f ,057 & ,054
zt ,044
~ ~ 1 std. dev. 5.6 5.5 3.8 4.7 5.8
uniform distribution of the 10 to 20 mg. over the cavity area. This generally requires 1 minute in the Wig-L-Bug. I n addition to this it is necessary to achieve a random distribution of all atoms so that the quantities AT) and p will adequately indicate the environment of each element. Starting from a mixture of standards components and grinding for 1, 2, 3, 4, and 5 minutes it was found that there was no change in observed concentration after the first minute. The samples ground for only 1 minute analyzed 1 to 6% high. The limitation which this effect might impose upon analysis of tissue slices was checked using a lyophilized rat lung. The general agreement between powder and slice are shown in Table Is’.
fl
26 24
n
22L
?
20
/
0
/
I 6 c
I2t
/
Figure 1.
Plot of cpo vs. absorbance ratio for phosphorus VOL. 37, NO. 13, DECEMBER 1965
1673
.
~~
Table IV.
~~
Analysis of Rat Lung, Powder vs. Slice“
Fe
Ca
Per cent in dry tissue K C1
0 032 1.14 Powder 0.087 0 033 1.19 Slice, 1 mm. 0.089 a Taken from adjacent positions within the rat lung. Table V.
3.360
KKa 3.744
S
P
1 12 1.12
1.33 1.39
Analysis of Rat Hippocampus, Dry Powder
W CaKa
1.29 1.26
= 13.8 mg.
B 4.03
XT~O= 1598.5 C.P.S. SKa: 4.729 5.373
C1 K a
TiKa(2) 2.275
PKa: 6.155
Counts per second
BR Bs BT~
.v
70
tsec.
0.500 0.500 2.45 1.92 0.76 0 8 . 4 1 431.3 o.oi9 1.88 50 180
0.500 1.73 0
...
... 240
0.500 0.95 0.29 12.06 0.56 180
Although this agreement is quite good the possible effect of a heterogeneous distribution of an element within a tissue slice must be kept in mind when extending the method to this application. The possibility of losing sulfur from the tissue during the irradiation process needed for analysis was considered and checked in the following manner. A standard composed of cystine, glycine, and potassium phthalimide was analyzed as is, after irradiation with 250-kv. x-rays (5800 r.) and after irradiation with Co60 (1 X lo6 r.). The signals from K K a and S K a as well as the observed values for AT^ were constant to within 10.9%. Similar results are obtained by repeated analysis of biological samples. The equation used previously (1) for the determination of tissue levels of Ca, K, C1, S, and P had the general form of Equation 7 but differed in the term used for correcting for interposing absorption edges. The absorption term constant of the earlier equation permitted fairly accurate analysis (=!=5%) over a limited range of concentrations such as are found in liver, lung, brain, etc. This was largely due to the relative constancy of the P term (=!=&lo%) for these tissues. For greatly different materials such as sulfolipids, phospholipids, red blood cells, etc., special sets of standards were necessary. The present equation adequately handles all of these materials with one set of standards. For example a prepared sample of choline sulfate (Theor. S = 17.5%) analyzed to be 17.3% S, and pooled samples of human red blood cells analyzed to be 0.286% Fe, 0.838% S, and 0.151% P in rather good agreement with the average values of 0.281% Fe, 0.896% S, and 0.154% derived from data tabulated by Bowen ( 2 ) . Because this scheme does not employ pulse height discrimination or make use 1674
ANALYTICAL CHEMISTRY
0,500 0.77 0.86 6.57 0.80 220
0.500 0.75 0 665.3
...
50
0.500 0.70 0.32 4.76 1.56 240
of a crystal particularly suited to discrim inate against higher order (n = 2, n = 3, or n = 4) reflections the magnitude of such high order interference must be considered. Two such interferences would normally occur in biological tissuesLe., Ca KP (n = 2) and Cu K a (n = 4) on P K a . The magnitude of the Ca Kp interference is 0.00032 X Ca K a signal which for the average sample requires a level of 1.3% Ca to raise the background at P K a by 10%. This correction need not be considered except for highly calcified tissue approximating bone. For the majority of tissues the copper level is so low (less than 0.002%) that Cu K a (n = 4) causes no interference. However, in the case of a hemocyanin system such as is found in lobster, a significant interference will exist. For dry lobster liver with a copper concentration of 0.24% the interference is equivalent to 7% of the total P K a signal or is 0.046 X Cu KCY(n = 2). The magnitude of these corrections is not normally large and may be kept to a minimum by using relatively small samples-Le., 10 to 15 mg. For particularly high calcium or copper value, together with relatively low phosphorus signals, a pulse height analyzing system must be employed. A typical set of values determined by this analytical method is shown in Table V. The reproducibility of these results is within =t3% for repeated counting of a single sample and 1 4 % for replicate analysis of the same sample. The approximate length of time required to count each signal is shown in the last row of the table. The greatest limitation to the speed of the method is the amount of time required to count S K a and P K a a t concentration levels of 0.1-0.2%. The net P K O signal in human red blood cells, for example, is 0.34 c.p.s. (approx. 0.15% P). Such a determination requires a count time of 20 to 30 minutes
to maintain a relative standard deviation of 3 to 12%. However, the equipment used in the development of this method is far from optimum when compared to the more conventional equipment used for the excitation and determination of soft x-ray intensities. Consequently it is easy to imagine a rather marked improvement in analytical speed by optimizing several of the basic components-e.g., use of an excitation source more suitable for exciting soft x-rays, operation of the source at constant potential and more nearly at maximum power, use of a diffracting crystal selected for its low wavelength efficiency, etc. (3) I n outline the analytical procedure is as follows: Weigh and lyophilize the fresh tissue. From the dry weight determine the per cent HzO. Grind the tissue for 1 minute in a Mixer-Mill to obtain a 25-mg. aliquot. Grind this aliquot in a Wig-L-Bug for 2 minutes to recover 10 to 20 mg. of fine powder. Distribute and smooth this powder into the 0.5- X 0.5- X 0.0037-inch cavity in a titanium planchet. Determine the weight of this sample. Determine the intensities from Ca K a , K K a , C1 K a , S K a , P K a , 4.03 A. and Ti K a (n = 2). Use Equation 4 to determine AT,. Use Equation 7 to determine individual concentrations. The sensitivity constants p t can be determined from suitable organic compounds such as calcium lactate, potassium phthalimide, chlorobenzoic acid, cystine, phosphoserine, etc., diluted with an organic matrix. Where the count rate is low enough the constant may be determined from the pure compound. ACKNOWLEDGMENT
The author thanks Shirley Wilt and John Campbell for their assistance in developing this procedure. LITERATURE CITED
(1) Alexander, G. V., A p p l . Spectry. 18, 1 - fl964). (2) Bowen, H. J. &I., “The Elementary \ _ _ _ _ ,
Composition of Mammalian Blood,” Re t . AERE-R4196 (1963). (3) ampbell, W. J., Brown, J. D., ANAL.
E
CHEM.36, 312 R (1964). (4) Doughman, W. R., Sullivan, A. P., Hirt, R. C., Zbid., 30, 1924 (1958). ( 5 ) Jones, R. C., Ibid., 33, 71 (1961). (6) Liebhafsky, H. A., Pfeiffer, H. G., Winslow, E. H., Zemany, P. D., “X-Ray Absorption and Emission in Analytical Chemistry,” p. 314, Wiley, New York, 1 96n.
(7) Natelson, S., Sheid, B., Clin. Chem. 6, 299 (1960). (8) Shreiber, T. P., Ottolini, A. C., Johnson, J. L., A p p l . Spectry. 17, 17 (1963). RECEIVEDfor review May 10, 1965. Accepted September 21, 1965. Work supported by Contract AT(04-I)GEN-IP
between the Atomic Energy Commission and the University of California.
