precision of the method. Table VI1 shows the mean concentration of each element detected and the corresponding relative standard deviation for the 13 elements detected in this platinum sponge lot. The over-all precision was 10% exl,ressetl as relative standard deviation. While no determination was made on the accuracy of the method, i t mas assumed to be qimilar to the precision.
ACKNOWLEDGMENT
The authors are indebted to H. Robinson s n d R. l l r d y u s for their
preparation of the many standards required in this study. LITERATURE CITED
(1) Churchill, J. R., IND.EN^. CHEM., ANAL.ED. 16, 653 (1944). (2) Duffendack, 0. w., Wolfe, R. A., 10, 161 (1938). (3) Hammaker, E. M., Pope, G. W., Ishidaan, G., Wagner, F., Appl. Spectr. 12, 161 (1958). (4) Hawley, J. E., Wark, W. J., Lewis, C. L., Ott, W. L., Trans. Can. Zmt. Mining M r t . 54, 425 (1951). (5) Hodgkin, C. R., Hanson, J., Esso Research and Engineering Co., Linden, S . J., private communication (1961). (6) Pierce, W. C., Nachtrieb, N. J., IND. END.CHEM.,ANAL.ED. 13, 744 (1941). ( i )Raper, A. R., Withers, D. F., “Collec-
ted Papers on Metallurgical Analysis by the Spectrograph,” p. 144, Brit. NonFerrous Metals, Research h o c . , 1945. (8) Rupp, R. L.,Klecak, G. L., Morrison, G. H., ANAL.C H E M32, . 931 (1960). (9) Shaw, D. M., Wichremaainghe, O., Yip, C., Spectrochim. Acta. 13, 197 (1958). (10) Shllwood, B. J.1 J . Opt. SoC. Am. 44, 171 (1954). (11) Thierst R. E., APPl. SPectroscoPY 7, 157 (1953). (12) Twyman, F.,“Metal Spectrosco y ” p. 447, Charles Griffin, and Co. l!td,. London (1951). (13) Wark, W. J., J. Opt. Soc. Am. 41, 465 (1951). RECEIVEDfor review May 10, 1962. Accepted July 12, 1962. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 1962.
X-Ray Fluorescent Determination of Organic Substances through Inorganic Association G. J. PAPARIELLO, HERBERT LETTERMAN, and W. J. MADER Research Department, Cl8A Pharmaceutical Co., Summit, N. 1.
b An x-ray fluorescent method for the determination of organic compounds through inorganic association is presented, with a discussion of the properties that must be exhibited by the association reaction if it is to be suitable for use in this method. Three procedures of associating an x-raydetectable element with an organic compound are explored: bromination, chelation, and salt formation. Procedures for determining phenolic and unsaturated compounds by bromination, 5-chloro-7-iodo-8-quinolinol by chelation, and ammonium acetate by salt formation are outlined. This indirect method offers selectivity, specificity, and sensitivity.
T
HE use of x-ray fluorescent methods for the analysis of inorganic systems has rapidly advanced within the last ten years, and improvements are slowly but steadily being made in the determination of the “light elements’’ (sodium to titanium). On the other hand, the direct x-ray fluorescent analysis of organic systems, which would be most useful to the organic analyst, does not look promising at present because of the minimal fluorescent yield of organic compounds. Jackwerth and Kloppenburg (3) suggested the use of an indirect method of organic analysis by x-ray fluorescence at a time when this n-ork was already in progress; however, these authors did not explore the possibility but merely suggested its feasibility in conjunction with quantitative paper chromatography.
This indirect method might be likened to absorption spectrophotometric methods and lower energy (visible and ultraviolet) fluorescent methods, where pretreatment of the sample is required t o obtain a chromophor or induce fluorescence. The pretreatment used here is actually an association reaction between an organic and an inorganic substance. There are, of course, many ways by which a n organic substance could be associated with a detectable inorganic element; however, the association procedure which is chosen must meet several requirements. These requirements may be expressed most succinctly by referring t o the reaction of organic compound A with x-ray-detectable component B: A
+ nB F? AB, + (n - s ) B
(1)
The first requirement is that this association between A and B be such that the intensity measurements made a t a 28 angle of B be a quantitative reflection of the organic compound present as AB,. Secondly, there must be a simple quantitative procedure for separating product AB, from excess B without causing the equilibrium in Equation 1 to shift to the left. Three reactions which meet the above requirements were studied and are reported on a t this time: the bromination of phenolic and unsaturated compounds, the formation of a soluble, slightly ionized salt, and the formation of a n extractable chelate. The purpose of this work is not to present minute details on the determination of any one organic substance
but merely to illustrate the possibility of indirectly determining organic substances through their association with an x-ray-detectable element. The authors believed that a reaction would satisfy these requirements .if a definite, reproducible relationship between the amount of organic substance present and the measured radiation intensity could be established. This relationship was linear in all the cases considered. It would be necessary t o use a calibration curve, if a linear relationship was not obtained. EXPERIMENTAL
Association Methods. BROMINAA standard aqueous stock solution (0.01 t o 1 mg. per ml.) of the TION.
organic compound is prepared and from it a series of dilutions is made. Each solution in this series is made up t o a total volume of 20 ml. with distilled water. One milliliter of 6 N hydrochloric acid is added t o t h e test solution, followed by 5 ml. of bromine water, The solutions are permitted to stand for approximately 2 hours in the dark. They are then aerated for 1 hour. Each solution is transferred t o a 50-ml. volumetric flask. If the brominated product is insoluble, a solubilizing solvent such as N,N-dimethylformamide or ethyl alcohol is added and the solution is made up t o volume. A blank is prepared in a similar manner. CHELATION OF VIOFORM (5chloro-7iodo-8-quinolinol) . A standard stock solution (0.1 mg. per ml.) of the organic compound in a 50% water-acetone solvent is prepared. Using various aliquots of this stock solution (1 to 8 ml.), a series of dilutions is prepared in 250-ml. VOL 34, NO. 10, SEPTEMBER 1962
1251
CONCENTRATION (PARTS PER MILLION1
Figure 1. Relationship between guaiacol concentration and measured intensity of bromine line
separatory funnels. All the solutions are made up t o a total volume of 8 ml. with the acetone-water mixture. Then 50 ml. of a cupric nitrate solution (0.05 gram per ml. of solution using 0.04N hydrochloric acid as solvent) is added t o each. The separatory funnels are shaken for 2 minutes. The cupric chelate of Vioform is then extracted with four 10-nil. portions of chloroform. The chloroform extracts are collected in 50-ml. volumetric flasks and diluted to the mark with chloroform. A blank is prepared in a similar manner. A concentration technique is utilized at this point. -1section of hollow brass tubing 5 cm. in length and 2 em. in diameter is filled with asbestos paper tape and stood upright on a heated hot plate. A glass microscope slide is placed on top of this brass tube. Onemilliliter aliquots of the various solutions in the series, each in turn, are permitted t o fall dropwise onto a fresh slide, evaporation ensuing. The evaporation process is restricted within the area circumvented by the hot tubing. SALTFORMATION. A standard aqueous stock solution (0.25 and 0.0025 gram per ml.) of ammonium acetate is prepared and a series of dilutions is made up in 100-ml. volumetric flasks. Enough water is added to make the total volume in each flask equal to 50
Table 1.
Brominated Compounds and Range of Investigation
Lower
Compound Phenol Th mol m-eresol PHexylresorcinol Guaiacol Salicylic acid Diallylbarbituric acid
1252
e
Upper Limit, P.P.M. Dimethylform. ... ~
Limit, P.P.M. H?O 0.2 0.5 0.2
0.2 0.3 0.4
1.0
10 50 ... ,
~
amideH20
.
SO
30
450
ANALYTICAL CHEMISTRY
50 250 50
50
... .
, ,
,..
Figure 2. Relationship between Dial concentration and measured intensity of bromine line
RESULTS AND DISCUSSION ml. Exactly 3 grams of lead sulfate is then added to each solution. The Bromination. The bromination of solutions are mechanically shaken for 1 hour, left standing at room temperaa number of compounds was atture for 1 more hour, then diluted to tempted. Since the chemical state the mark. The excess lead sulfate is of a n element is not usually disremoved by filtering. A blank is tinguishable by x-ray fluorescent prepared in a similar manner. methods, it was necessary to remove X-Ray Analysis. INSTRUMENTAL the brominating agent prior to deCONDITIONS.T h e General Electric termination of the brominated prodXRD-5 x-ray fluorescent unit is used. uct. Bromine water was used as the The primary radiation is supplied by brominating agent rather than the a molybdenum target x-ray tube opusual bromide-bromate solut,ion beerated at 50 kvp. and 50 ma. A lithium fluoride analyzing crystal and cause of the facility with which the a gas proportional counter (General excess bromine could be removed by Electric No. 6 tube) are employed. aerating t h e reaction solution. The following goniometer settings are The bromination reaction was parused for the respective association ticularly attractive because the stoichimethods: ometry of the reaction favors greater sensitivity of the method. In the 20 case of the bromination of salicylic Method .4nalyzing Line Angle, O acid, for example, decarboxylation Bromination Bromine, K a first 29.97 occurs, and 2.4,6-tribrornophenoI is order obtained (4). Hence, for every one Chelation Copper, K a first 45.03 molecule of salicylic acid originally order present, there are three measurable Salt forma- Lead, La first 33 93 tion order bromine atoms. The addition of bromine t o a carbon double bond will likewise show favorable stoichioinetry, The scaler circuit is set to record the yielding two s-ray-detectable bromine number of counts accumulated in 100 atoms for every one unsaturated linkseconds. age. -411 instrument conditions are the Table I lists the compounds brosame for the three association methods, except for the 28 angle used, and the minated. As this table indicates, the inverted optics kit (Spex Industries), range of investigat,ion was on a partswhich is used for the liquid samples per-million level. The lower limit of obtained in the bromination and salt the method was imposed by the information procedures but not for the herent capabilities of x-ray fluoressample obtained from the chelation cence in determining bromine in the procedure. matrix of the method. The upper PROCEDURE. The series solutions limit mas restricted by the solubility which are the end products of the broof the bromihated product and hence mination and the salt formation procedures are placed in liquid sample could be increased. In certain cases cells, and each in its turn is placed in the upper limit was raised by use of a the sample cell drawer. I n the case of dimethylformaniide-water mixture as the concentrated sample on the glass solvent; however. no attempt 11-as slides obtained from the chelation promade to find the most, suit,able solvent cedure, the slide is positioned in the system. opening of the aluminum mask of the In the range investigated 3 linear sample drawer and the counts accumrelationship between organic compound ulated at the end of 100 seconds a t the concentration and measured radiation indicated 20 angle and under the aboveprescribed conditions are recorded. intensity was obtained for all t,lie com-
Table 11. Accuracy and Precision Obtainable in Determination of a Salicylic Acid Sample
(Theoretically 2.0 p.p.m.)
De-
termined
concen- Deviation, P.P.M.
Sam-
tple 1 2 3
Av.
tration, From P.P.M. theoretical 0.3 2.3 0.3 2.3 2.1 0.1 2.23 0.23
From mean 0.07 0.07 0.13 0.09
pounds listed in Table I. Figures 1 and 2 clearly indicate that the bromination of a phenolic and an unsaturated compound, guaiacol and Dial (diallylbarbituric acid), respectively, definitely lends itself to the indirect determination of these compounds by x-ray fluorescence. To obtain some conception of the accuracy and precision of the bromination procedure, salicylic acid was brominated, a calibration curve in the range of 0 to 10 p.p.m. was prepared, and three samples of salicylic acid, each made to contain 2 p.p.m. theoretically were assayed (Table 11). Only 10 ml. of solution is needed as a working sample in this procedure. Hence, it would be possible to obtain a precision to approximately *4% with as little as 20 fig. of salicylic acid sample. Chelation. The chelation and detection of only one compoundVioform-are reported, although other chelation procedures are being studied. T h e formation of a cupric chelate of
i
Vioform in acidic solution has been reported (1). After the chelate is formed under the prescribed conditions, the chelate is extracted into chloroform. The chelating agent, cupric nitrate, stays behind in the aqueous layer and hence there are no separation difficulties. A direct measurement on the chloroform extract would be unwise, since there is sufficient heat within the sample chamber to affect a volatile solvent such as chloroform. This obstacle along with the slight solubility of the chelate in the chloroform and a high background count, due to the presence of copper impurity in the molybdenum target of the x-ray tube, suggested the use of a concentrating technique. Consequently, the method of concentrating the sample on a glass slide was developed. There was difficulty in obtaining a uniformly deposited sample. A wide variation in readings was obtained by merely removing, reorienting, and replacing the slide in the sample drawer, as is illustrated in Figure 3. This error might he reduced considerably by the use of a turntable sample holder (Spex Industries No. 3520). Salt Formation. The determination of ammonium acetate through the formation of the soluble, slightly ionized lead acetate salt serves as a n example of how salt formation might be used in the indirect determination of organic compounds by x-ray fluorescence. The reaction (8) which makes the determination of ammonium acetate by this procedure possible is :
+
PbSOi 2”4+ Pb(CzHs02)z
+ 2CzHa02+ 2(”4)+ + SO,-’ 4
(2)
In this procedure, insoluble lead sulfate is placed in solution by the formation of lead acetate. Thus the amount of lead in solution is a direct reflection of the amount of acetate ion present. The insoluble excess lead salt is merely removed by filtration. A blank is necessary, since lead sulfate will exhibit some minimal water solubility. Figure 4 shows a linear relationship between ammonium acetate concentration and the measured radiation intensity over the range investigated. CONCLUSION
-
o
m.
PER W Y I .
06
Figure 3. Relationship between Vioform concentration and measured intensity of copper line
The three reactions which were successfully investigated are not unique-there are many more reactions between organic compounds and x-raydetectable components which will enable the indirect determination of organic compounds by x-ray fluores-
0
En
a4 CENT ACETATE
I
Figure 4. Relationship between acetate concentration and measured intensity of lead tine
cence. Thus it is not too difficult to envision the wide use of this method in quantitative functional group analysis. Work is a t present in progress in these laboratories to utilize such reactions as the chelation of amino acids, the chelation of pyridyl derivatives, and the reaction o f t e r t i a r y amines with methyl iodide to form quaternary ammonium salts in this indirect x-ray fluorescent method of organic analysis. This method provides the chemist with another tool and offers the advantages of specificity, selectivity, and sensitivity. Specificity and selectivity can be obtained by the proper choice of the inorganic association reaction. Thus in many cases lengthy, time - consuming, error - introducing separation procedures may be unnecessary. This method has the potential of assaying, without isolation, microgram quantities in highly colored, manycomponent solutions. Thus it should be particularly attractive to chemists confronted with the difficult determination of drugs and metabolites in body fluids. ACKNOWLEDGMENT
The authors thank Anton A. Benedetti-Pichler of Queens College for his advice and interest in this work. LITERATURE CITED
(1) Berg, R., 2. anorg. allgem. Chem. 204, ( 2208 ) Engelder, (1932). C. J., “Fundamentals of
Semi-Micro Qualitative Analysis,” p. 179, Wiley, New York, 1950. (3) Jackwerth, E., Klop enburg, R. G., Z. anal. Chem. 179, 188 (1961). (4) Ruderman, I. W., IND.ENQ. CHEM., ANAL.ED. 18,753 (1946). RECEIVEDfor review May 28, 1962. Accepted July 5, 1962.
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