bias toward low or high results was observed and a maximum relative error of 3.6% was obtained. These titrations were carried out using 4 : l dodecanolpyridine diluent. As shown in Table 111,the use of two high molecular weight alcohol-pyridine mixtures had general application to systems where methanol is known to cause serious interfering side reactions. I n many cases, the previously reported solvents and solvent mixtures are not as effective in eliminating or minimizing the side reactions. A maximum relative error of 7y0 was found. I n all these examples, except with vinyl ethers, the water reaction rate was normal and end points were easily obtained. With vinyl ethers, the rate of the water-KFR reaction was inhibited greatly. End points were difficult to observed with the rapid titration method. The data in Figire 5 suggest that a small excess of K F R should be taken in addition to the normal end point excess, the sample added, and about 3 to 4 minutes allowed for the water reaction. Then the titration may be completed to the initial current level of the excess reagent. Advantages using the new titration method were also found in determination of water in noninterfering samples. h comparison was made on precision and speed of titrations with a sample of water in methanol using the new method and
other end point detection devices commonly employed, including a n automatic titration unit. Increased speed of titrations (2 to 3 times faster) is possible with 2 to 6 times better precision using the new method. Titrations using the modified apparatus were carried out by a continuous addition of K F R at a rate of about 1 m1./5 seconds until levels were approximately matched. Small additions of K F R were then made to complete titration of the least traces of mater to a n end point of closely matched current levels. The method and apparatus described have been employed routinely in our laboratories by hourly technicians for about a year. They have proved to be valuable for rapid and precise determinations of water in the presence of interfering compounds. We also use the same end point method for KFR titrations of water in noninterfering materials dissolved in methanol. The apparatus required was simple to assemble, and the only major expense was the potentiometric recorder. LITERATURE CITED
(1) Barbi, G. B., Pizzini, S., ‘AKAL. CHEIII.35. CHEW 35, 309 11963). (1963). (2) Barnes, ’L., (2yB;rnes, L., Pawlakj Pawlak, 11. S., Zbid., 31,
1875 (1959). (3) Bastin, E. L., Siegel, H., Bullock, A. B., Zbid., p. 467.
(4) Burns, E. A., Muraca, R. F., Zbid., 34, 848 (1962). (5) Damm, K., Noll, W., Kolloid 2. 158, 97 11958). (6) Fischeg, K., Angew. Chem. 48, 394 f 1 9.?.5\ \ - - - - I .
(7) Gilman, H., Miller, L. S., J. A m , Chem. SOC.73, 2367 (1951). ( 8 ) Grubb. W. T., Zbid.. 76. 3408 (1954). (9) Hampton, J. ’F.,Lacefield, G. W:, Hyde, J. F., Znorg. Chem. 4, J659 (1965). (10) Kolthoff, I. AI., Elving, P. J., “Treatise on Analytical Chemistry,” Part TI/ Vol. 1 (Mitchell, J.), pp. 84-87, Interscience, New York, 1961. (11) Meyer, A. S., Boyd, C. XI., Zbid., 31, 215 (1959). (12) Mitchell, J., Smith, D. M., “Aquametry,” pp. 71-102, Interscience, New York, 1948. (13) Zbid., p. 104. (14) Zbid., pp. 155-156. (IS) Zbzd., p. 256. (16) AIurayama, M.,U. S.Patent 2,834,654 iMav 13. 1958). (17) Peter;, E. D.,‘ Jungnickel, J. L., ANAL.CHEM.27, 450 (1955). (18) Potter, E. C., White, J. F., J . Appl. Chem. 7, 309 (1957). (19) Noll, W., Damm, K., Krauss, W., Farbe Lack 65, 17 (1959). (20) Stock, J. T., “Amperometric Titrations,” pp. 166-174, Interscience, New Ynrk 1965. -----I
(21) Zbid., pp. 54-55. (22) Swensen, R. F., Keyworth, D. A., ANAL.CHEX 35, 863 (1963). RECEIVEDfor review July 26, 1965. Accepted October 22, 1965. 13th Anachem Conference, Wayne State University, Detroit, Mich., October 1965.
Pyrolysis f o r St ruc t ure Dete rmina t io n of Cyclopropene and Cyclopropene Fatty Acids JOANNE L. GELLERMAN and HERMANN SCHLENK University of Minnesota, The Hormel Institute, Austin, Minn. Pyrolysis of cyclopropane fatty esters at 350” C. with Si02 as catalyst yields numerous olefinic isomers. The structure of the original ester can be concluded from the products of ozonization-hydrogenation of the olefinic mixture. The same method has been applied to sterculic ester. The cyclopropene ring requires 160” C. with Si02 for cleavage. Dienoic esters result and the products of oxidative fragmentation are again characteristic for the original structure. Hydrogenation with Pd-Pb catalyst in methyl acetate reduces sterculic ester selectively to dihydrosterculic ester while common unsaturated fatty esters remain unchanged. The latter are reduced selectively by PtOz in methanol without concurrent hydrogenolysis of the cyclopropane ring. Proper combination of hydrogenations with pyrolysis enables determining the structure of a cyclic acid without isolating it in purity.
72
ANALYTICAL CHEMISTRY
L
FATTY ACIDS containing a cyclopropane or cyclopropene ring are of interest as constituents of certain bacterial and plant lipids ( 1 , IS). Methods for identification of their structure-i.e., for locating the position of the ring-methylene group in the chain-involve chemical reactions which lead eventually to branched and/or normal chain esters. These are then subjected to any of the conventional oxidation methods for identifying the position of a side methyl group, double bond, or keto group in fatty acids (6, 7 , ONG-CHAIN
Id).
The procedures reported here are based on pyrolytic cleavage of the ring followed by ozonization-hydrogenation of the resulting mixture of unsaturated compounds. The methods may hold some advantage above others in regard to time, simplicity, and amount needed. I n gas-liquid chromatography (GLC) of cyclopropane fatty acid methyl esters, certain phase supports give rise
to several peaks which indicate decomposition of the ring ( 8 ) . Catalysts, temperatures, and periods of heating were studied to develop a controlled method for ring fission which is independent of GLC. Silicic acid as used for adsorption chromatography catalyzes the reaction in a sealed tube a t 350” C. very satisfactorily. A mixture of mainly monoenoic compounds results and only minimal amounts of polymers are formed. In pyrolysis of a cyclopropene ester-Le. , methyl sterculate-heating with silicic acid for 15 minutes a t 160’ C. is sufficient and the recoveries are as satisfactory as \Tith cyclopropanes. The dienoic products from sterculate resemble those which Kircher and coworkers (20) obtained under quite different conditions from the corresponding hydrocarbon, sterculene. Cyclopropane and cyclopropene fatty esters must not necessarily be isolated in purity to apply the pyrolysis method.
Normal saturated long-chain fatty esters do not interfere with pyrolysis and their misinterpretation as products from oxidation can be avoided. However, unsaturated fatty esters in the original sample make the conclusions from oxidation fragments ambiguous. Therefore, in determining the structure of cyclopropane fatty esters, admixed olefinic esters must be selectively reduced without hydrogenolysis of the cyclopropane ( 7 ) before pyrolyzing the latter. On the other hand, it was found that the cycloprogene ring can be hydrogenated selectively. Pd on CaC03, modified with P b salt (9), is known to catalyze hydrogenation of acetylenic compounds to cis olefines in very satisfactory yields. This catalyst serves here the purpqse of reducing the double bond in the ring without hydrogenolysis while concurrent hydrogenation of normal olefinic esters takes place only to a very minor extent. By such selective reduction, sterculic ester becomes amenable to the procedure outlined for cyclopropane esters. This route of elucidating in mixtures the structure of cyclopropene fatty esters via cyclopropane compounds may sometimes be of value in view of their notorious tendency to autoxidize, polymerize, or isomerize in uncontrolled fashion. However, in omitting isolation of the cyclic ester, one has to be aware that other cyclic or branched esters might interfere when present in appreciable amount. EXPERIMENTAL
Gas-Liquid Chromatography. Retention data are given as equivalent chain length (ECL) ( 1 1 ) . Conditions for GLC used in this laboratory have been specified elswhere (4, 14, 16-18). Isomeric fatty esters with a methyl branch or a double bond in different positions of the center area of the chain
were not separated. Similarly, it is known that cyclopropane esters are hardly separable from corresponding monoenes-e.g., lactobacillic from 19: 1 -and cyclopropene esters from dienese.g., malvalic from 18:2 (23). It was also found with authentic compounds that methylketones were not separated from the isomeric straight-chain aldehydes. Hydrogenation of Fatty Esters. The modified Pd catalyst was prepared according to Lindlar (9). Hydrogenations were carried out in a Parr apparatus or in a test tube a t atmospheric pressure with hydrogen bubbling through the solution. Table I lists the hydrogenation conditions and pertinent results of tests. I n contrast to the highly selective reactions, it was found that hydrogenation procedure I1 yields from sterculate about 70YGdihydrosterculate and 30% of a mixture of normal and branched esters, although cyclopropane compounds are hardly attacked under the same conditions. It was a150 attempted to achieve the results of hydrogenations I and I1 in one step by use of palladium catalysts. However, the amount of branched compounds from sterculate was larger and the results were not as consistent as with consecutive selective hydrogenations. Ozonization. Ozonization and subsequent reduction of ozonides to carbonyl compounds were carried out as described elsewhere ( 4 , 14, 16). .ildehydes and 2-heptanone, 2-nonanone, 2-undecanonej and 2-tridecanone were purchased (K & K Laboratories) for GLC reference. ,Ildehydes and aldehyde esters were also identified by reference to the ozonization-hydrogenation products of authentic unsaturated fatty esters. 12-Ketostearate was prepared from hydrogenated ricinoleate ( 2 ) for comparison with some of the ozonization products from pyrolyzed compounds, Alodel compounds for other possible ozonization products were not available.
Table 1. Procedure NO.
I
Solvent
Catalyst
Methyl acetate
Pd-Pb
Sample/ catalyst,
Fatty Acids and Esters. Common straight-chain fatty esters were obtained from The Hormel Foundation. Several methyl branched fatty esters were available from previous syntheses (17) and unsaturated straight-chain C19 esters had been isolated from fish oil (19). 9,lO - hfethylenehexadecanoate was synthesized from palmitoleate according to Simmons and Smith (21). 9,lOand 6,7-methyleneoctadecanoate were prepared from oleate and petroselinate by the same method. The crude products were purified by crystallization after hydrogenation I1 of residual olefinic ester. According to GLC, the preparations were at least 95% pure with palmitate and stearate being the respective contaminants. Infrared spectra showed the absorption band a t 9.8 microns ahich is characteristic for the cyclopropane ring. Fatty acid mixtures containing cyclopropane acids were obtained from lipids of Enterobacter cloacae and they were studied as concentrates ( 8 ) . Sterculia foetida was the source for 01- (2-n-octylcycloprop-1-ethyl)-octanoic acid (9,1O-methylene-g-octadecenoic, sterculic). The crude acids from alkaline saponification of the oil were subjected to crystallization a t -17" C., 20 grams in 200 ml. of acetone. The mother liquor was cooled to -50" C. and yielded 10.6 grams of a precipitate which consisted of 88% sterculic, 3y0 palmitic, and 9% oleic acids according to GLC in combination with hydrogenation I and 11. The concentrate shelved the proper I R ahiorption a t 5.35 microns (weak) and 9.92 microns (strong). It was stored under SBa t - 18" C. After 2 months the spectrum n-as the same and absorption a t 6.09 microns which would indicate deterioration (3) was not detectable. The acids were esterified in 2-gram portions. A11 esterifications of cyclic acids were carried out a i t h CHzSz (4, 15). BF,methanol which is often used as catalyst for esterification on small scale
Hydrogenation Conditions
w./w.
Pressure
Time
between 1 : 2 and
atm.
5 min.
Hydrogenation or hydrogenolysis of
Recovered i n tests
C
C
C
/\
/\
/\
C
C
C=C; but not
1:lO
95% C-C from C-C; >95ycoleate from oleate
C
/\
c-C,
I1
CHSOH
PtOz
1:1
40 lbs.
1 hr.
C=C
Ca
d>,C=C;
but not
98% C-C /\ from C-C; /\
oleate
C
-+
stearate
/\
C-C
I11
@
Acetic acid
PtO,
1:l
40 Ibs.
1 hr.
C C=C;
/\
c-c
C
A
C-C
-+
n
+ br
The course of hydrogenation is not uniform, see text.
VOL. 38, NO. 1, JANUARY 1966
b
73
Table II. GLC Peaks from Pyrolyzed 9,lO-Methylenehexadecanoate ECL on Peak p-CDX ECLon Area, No. Val.= EGS % A 16.15 16.3 B 16.42 16.9 C 16.73 17.2 D 17.00 17.6 p-Cyclodextrin valerate ( 1 7 ) phase of low polarity. (1
29 18 24 29
is a
(10) completely destroys the cyclopropane ring, partly involving methoxylation. The destructive effect of strong acids-e.g., HC1 in methanol on the cyclopropane ring-is well known (82'). Pyrolysis. Between 5 and 100 mg. of cyclic ester or saturated common fatty esters containing them were mixed under Nzwith twice the weight of silicic acid as commercially received (Mallinckrodt A.R., 100 mesh), in tubes 8 mm. 0.d. and 20 cm. long, one end sealed. The tubes were sealed under I\T2 a t atmospheric pressure to obtain ampules about 7 cm. long. They were completely immersed in smoothly fitting holes of a preheated aluminum block, 12 X 18 X 6 cm. The results of pyrolyses were checked for disappearance of cyclic compounds by appropriate hydrogenation and subsequent GLC. Cyclopropane fatty acids or esters are pyrolyzed to >95% Fvith silicic acid as catalyst by heating for 30 minutes a t 350" C. Sterculic acid was pyrolyzed in ester form and required with silicic acid catalyst only 15 minutes a t 160"C. for virtually complete conversion. Recoveries were >90% from 20- to 100-mg. samples. I n several experiments it was indicated that recoveries are slightly lower, 85-90y0, when 100- to 200-mg. samples are used. The possibility of preparing branched from these cyclic compounds by pyrolysis on larger scale was not explored. Unwashed supports for GLC derived from diatomaceous earth were found similarly active. Decomposition of the cyclopropane ring was also catalyzed by alumina but it was incomplete under comparable conditions. Isomerization takes place in liquid phase. Refluxing a t 350" C. in an ampule which is not completely immersed into the heating block is bound to give incomplete conversion. Heating a t respective temperatures without any catalyst rendered essentially unchanged starting materials from cyclopropane as well as from sterculic esters. Formation of Acids under Pyrolysis Conditions. Thin layer chromatography of pyrolyzed mixtures from cyclopropane f a t t y esters showed t h a t they were saponified t o a n extent of about 50%. The same effect was observed when commercial unwashed GLC supports were used as catalyst. Especially washed preparations of diatomaceous earth still saponified esters a t 350" C. but did not isomerize the cyclopropane ring. These observations are of bearing in GLC of the cyclic
-
74
ANALYTICAL CHEMISTRY
esters and possibly other applications of GLC a t high temperature. After pyrolysis a t 350" C. the materials were esterified or re-esterified with CH2N2. No difference was found in isomerization products from acids and esters of cyclopropane type so that esterification for the pyrolytic reaction is unnecessary and may merely be a n additional step which must be repeated afterwards, Saponification takes place only in negligible amount with sterculate under the milder conditions of pyrolysis. Reesterification was superfluous. RESULTS A N D DISCUSSION
Cyclopropane Esters. IR absorption a t 10.37 microns showed, in reference to elaidate, t h a t about one-third of the mixture from cyclopropane esters had become trans olefine. I n further procedures, 9,lO-methylenehexadecanoate was studied most extensively so t h a t it is the example in the following considerations. Pyrolysis yields a mixture of monoenoic C17 compounds which after esterification and hydrogenation 11 gives two peaks in GLC. One of these peaks represents n-C17 ester, the other corresponds to C17 esters with a methyl group in the center area of the chain. GLC of the nonhydrogenated pyrolysis products gives four well separated peaks, A-D, which are characterized in Table I1 and exemplified in Figure 1. The products of ozonization-hydrogenation obtained from the total mixture yield a rather simple pattern in appropriate GLC procedures (4,14,16). Four peaks correspond to C S C ~ aldehydes and four peaks correspond to CgCll aldehyde esters. It will be shown afterwards that these peaks represent, a t least in part, straight-chain aldehydes and aldehyde esters. The shortest aldehyde and aldehyde ester are significant for the position of the original cyclopropane ring. The ring is that part of the chain which is between these fragments-Le., the structure of the ester selected here is H8C(CHJ5-CaH4-(CH2)7COOMe, 9,lO-methylenehexadecanoate. Pyrolysis of the cyclopropane ring involves only the C atoms directly adjacent to the ring. Theoretically, the isomers listed in Table I11 may be foreseen and it is likely that each of them occurs in detectable amount. Fractions were collected from GLC (18) and materials sufficient for ozonization were obtained from peaks A, C, and D (Figure 1). GLC of the hydrogenated fraction D showed that it consists solely of n-CI, esters. Ozonizationhydrogenation of D yielded C8-C9 aldehydes and C&,, aldehyde esters. Accordingly, peak D represents isomers 1-4 (Table 111) and their fragments are straight-chain compounds. They must contribute to the respective peaks in
ci4 ci6 Ci8 Figure 1. GLC of pyrolyzed 9,lOrnethylenehexadecanoate on p-cyclodextrin valerate the chromatograms of the ozonized total mixture. GLC analysis of fraction d showed only one peak besides some palmitate which had been present in the original mixture. Fractions B and C, however, turned out to be mutually contaminated. From ozonization of h and C, under consideration of relative amounts, i t is likely that isomers 7 and 8 are fraction A, while isomers 9-12 are in fractions B and C. A peak, ECL 23.0, was found after ozonization of fraction C, and it represents longchain keto esters from isomers 5 and 6. This peak occurred also in GLC when the total mixture had been ozonized. The assignment of ECL 23.0 to ketopalmitate is supported by 12-ketostearate having ECL 25.0, and by a peak, ECL 25.0, which arises by same procedures from 9,10-methyleneoctadecanoate. The above assignments of isomers to peaks A-D are, in part, only tentative. It appears, however, that isomers combining a double bond and methyl branch yield easier to GLC separation than isomers having only one of these features. The same may pertain to the configuration of the double bond when close to a methyl branch. Structure determination of cyclopropane fatty esters is independent of assigning the branched unsaturated structures to specific ECL. It is also independent of identifying all of the oxidation fragments. Proof has been given for the presence of straight-chain fragments and from Table 111 the shortest aldehyde and aldehyde ester are expected only as straight-chain compounds. The position of the cyclopropane ring is concluded from them. Experiments with 9,lO- and 6,7methyleneoctadecanoate gave corresponding results. The former yielded peaks of, and equivalent to, aldehydes Cs-Cn and aldehyde esters Cg-C11; the latter gave peaks for C d L aldehydes and CSCs aldehyde esters. Again, the correct structures are concluded from the shortest fragments.
+
alumina for 35 hours a t room temperathe isomer equivalent to 5 6 was The method was also applied to fatty ture. The products were distilled and present only in very small amount. esters from Enterobacter cloacae where The results obtained here from stercuthe structures of the distillate comthe presence of 300/, cyclopropane comlate are well compatible with the scheme ponents determined. About 60% of pounds had been detected by I R isomer corresponding to 1 2 were and the quantifications by the earlier spectrophotometry. GLC of the mixfound, 300/, equivalent to 3 4, but authors. ture after hydrogenation I11 verified this and, in combination with hydrogenation 11, indicated the presence of %Yo cyclic CIi and of 5% cyclic C l ~ compounds. However, the position of Table 111. Possible Isomers from Pyrolysis of 9,lO-Methylenehexadecanoate and the ring had to be established in acids Their Oxidation Products from this novel source. After enrichment according to chain length, 9,lOA: CHa(CHz)sB: - - ( c H ~ ) ~ c o o M e Isomer Aldehyde Ald-ester methylenehexadecanoic and lactobacillic acids were identified by C / \ pyrolysis and ozonization (8). 1 A-C-C C=C-B c 9 CS Dihydrosterculate \vas prepared from ,-. sterculate by hydrogenation I, and /-\ contaminating oleate was reduced by 2 -c-c c-cCS c 9 hydrogenation 11. The mixture of 88% C dihydrosterculate, 9% stearate, and 3y0 palmitate gave, upon pyrolysis, re3 -c-c / \c-cCl ClO sults as expected from synthetic 9,lOC methyleneoctadecanoate with these contaminants. Pyrolysis of sterculate itself 4 -c=c / \c-cC6 c 1 1 is discussed in the following. C Sterculate. The ultraviolet specII CH20,Keto-ester 5 -c-c-c-ctrum of pyrolyzed sterculate had strong absorption a t 231 mk which C corresponds to conjugated diene. XsII CH20, Keto-ester 6 -C-C-C-Csuming a molar extinction coefficient of C 22,000 (6, do), nearly 80% of the sterculate had been converted into such com7 -c=c-L-cbr-Cl1 C6 pounds. The I R spectrum of the mixC ture showed bands a t 6.05, 6.18, and a t I 10.37 microns indicating 45gib trans Cs-br 8 -C-C-C=CC8 double bond/mole; other absorption C was found a t 11.37 microns with a 1 slight shoulder a t 11.12 microns. 10-keto-Cll 9 -c=c-c-cC6 After rearrangement of sterculate and C hydrogenation 11, GLC of the products 2-keto-C8 10 -c-Lc-cCP showed that only between 3 and 7.5% straight-chain esters have been formed C I while they represent about '/3 of all prod8-keto-Clo 11 -c-c=c-cc 7 ucts from cyclopropane ester. OzonizaC tion-hydrogenation of the pyrolyzed I mixture gave GLC peaks corresponding 2-keto-C 12 -c-c-c=cCS to 5.5% Ci, 72.9% Cs, 13.2% Cg, 8.1% Clo aldehydes and to 5.1% Ci, 79.7% CS, 8.6% C9, 6.6% Cl0 aldehyde esters. However, the sample of sterculate conTable IV. Isomers from Pyrolysis of Sterculate (9,1O-Methylene-9-Octadecanoate) tained 9% oleate which is close to the and Their Oxidation Products values found for Cg aldehyde and Cg A: CHa(CH&B: -(CH&COOMe aldehydes ester. Furthermore, 3% of Isomer Aldehyde Ald-ester methyl palmitate contaminant is pres,-. L ent which superimposes with CS alde11 hyde ester, but its contribution to the ? 1 A-C-C-C-C=C-C-B CS peak area is insignificant. CSfragments C outrank the other products by a factor I1 2 -c-c=c-c-c-cCS ? of nearly 10. The position of the original cyclopropane ring can be conC cluded from these major products as they represent, together with the ring, the whole carbon structure of sterculic acid. When the scheme of rearrangements derived from sterculene by Kircher and coworkers (90) is adapted to sterculate, one has to expect the isomers listed in Table IV. These authors rearranged, on much larger scale, sterculene in petroleum ether by treatment with
+ +
VOL. 38, NO. 1, JANUARY 1966
75
Cscompounds are crucial for structure determination and they arise from isomers 1-4 in abundance when compared with those from 5 to 6. The fate of fragments complementary to Cs from isomers 1 and 2 was not investigated under our conditions. The di-functional products which may arise from diozonides do not interfere with GLC of the mono-aldehydic fragments. However, a large amount and great variety of unsaturated straight-chain esters together with a cyclopropene compound would make stepwise hydrogenation before pyrolysis the more reliable course of procedures. ACKNOWLEDGMENT
Thanks are due to N. Larson for lipids of Enterobacter cloacae, M. Guerrero for Sterculiafoetida seeds, J. R. Chipault and W. Deutsch for IR spectra and their discussion. Laboratory assistance by P. Eklund and E. A. Quam.
LITERATURE CITED
(1) Carter, F. L., Frampton, Chem. Rev. 64, 497 (1964).
V. L.,
(2) Christie, W. W., Gunstone, F. D., Prentice, H. G., J . Chem. SOC.1963, p. 5768. (3) Faure, P. K., Smith, J. C., Zbid., 1956. D. 1818. (4) Geilerman, J. L., Schlenk, H., J . Protozool. 12, 178 (1965). (5) Gillam, A. E., Stern, F. S., “An Introduction to Electronic Absorption Spectroscopy in Organic Chemistry,” 2nd ed., p. 93, E. Arnold Ltd., London, 1957. (6) Hofmann, K., Marco, G. J., Jeffrey, G. A., J . Am. Chem. SOC.80, 5717 (1958). (7) Kaneshiro, T., Marr, A. G., J . Biol. Chem. 236, 2615 (1961). (8) Larson, K. L., Gellerman, J. ,L., Abstract Xo. 119, 65th Annual Meeting, American Society hlicrobiology, Atlantic City, April 1965. (9) Lindlar, H., Helv. Chim. Acta 35, 446 (1952). (10) Metcalf, L. D., Schmitz, A. A,, AXAL. CHEM.33, 363 (1961). (11) Miwa, T. K., Mikolajczak, K. L., Earle, F. R., Wolff, I. A., Ibid., 32, 1739 (1960).
(12) Nunn, J. R., J . Chem. SOC.1952, D . 313. (13) O’Leary, W. M., Bacteriol. Rev. 26, 421 (1962). (14) Sand, D. M Sen, N., Schlenk, H., J . Am. Oil C h e m h ’ SOC.42,511 (1965). (15) Schlenk, H., Gellerman, J. L., ANAL.CHEM.32. 1412 (1960). (16) Schlenk, H., ’ Gellerman,’ J. L., J . Am. Oil Chemists’ SOC.42, 504 (1965). (17) Schlenk, H., Gellerman, J. L., Sand, D. M., ANAL.CHEM.34. 1529 (1962). (18) Schlenk, H., Sand, D. M., Ibid., p. 1676. (19) Sen, N., Schlenk, H., J . Am. Oil Chemists’ SOC.41, 241 (1964). (20) Shimadate, T., Kircher, H. W., Berry, J. W., Deutschmann, A. J., Jr., J . Org. Chem. 29,485 (1964). (21) Simmons, H. E., Smith, R. D., J . Am. Chem. SOC.81, 4256 (1959). (22) Thorne, K. J. T., Kodicek, E., Biochem. Biophys. Acta 59, 306 (1962). (23) Wolff, I. A., Miwa, T. K J . Am. Oil Chemists’ SOC.42, 208 (1985). RECEIVED for review September 7, 1965. Accepted October 25, 1965. Work supported by U. s. Public Health Service Grant AM 05165 and by the Hormel Foundation.
X-Ray Quantitative Analysis by an EmissionTra nsmissio n M etho d JEAN LEROUX and MAZHAR MAHMUD Occupational Health Division, Department of National Health and Welfare, Ottawa, Canada
b This method was developed to permit quantitative determination of elements with acceptable accuracy, in most cases without the use of time-consuming methods involving internal standards, calibration curves, etc. The theoretical approach is described, with the experimental conditions which yielded a general equation expressing the concentration, C,, of a given element in a tested sample. The “matrix effect” being experimentally taken care of by the transmission measurements introduced in the general equation, the method has been proved accurate with samples including ranges of extreme values of linear absorption coefficients. The method applies to any type of sample, whatever its physical state, if the element to be tested is hornogeneously dispersed in the sample and the sample does not absorb too much of the analyzed radiation. Means of fulfilling these conditions are discussed.
M
samples submitted to this laboratory for analysis vary in physical condition and chemical composition. They usually come as dusts, powders, lumps of rock, liquids, and metallic samples. Among the tech76
niques and equipment used, the x-ray spectrograph was reserved chiefly for the analysis of powder samples. It was early realized that no general method could be used for a quick analysis of these samples. The difference in their composition was so large that each required tests and calibration curves by conventional methods because of the unavoidable problem of the matrix effect. Therefore, the use of the instrument had to be restricted to a limited field of applications unless direct experimental evaluation of the matrix effect could be made. The method described in this paper was then investigated, as it was inspired by a similar problem related to x-ray diffraction (6-8). The following theoretical approach, which is slightly different in its basic assumptions from those referred to by Birks (3) and Liebhafsky (9), has been derived to suit the specific experimental conditions imposed by the transmission measurements involved.
OST
ANALYTICAL CHEMISTRY
THEORETICAL APPROACH
Figure 1 s h o w the x-ray emission taking place from element i of concentration C, and homogeneously dis-
persed in the flat pellet of density p and thickness H . We call I , the portion of the continuous spectrum of the primary beam contributing to the emission of a given characteristic radiation of i. The wavelength range of this portion coincides mainly with the immediate short wavelength side of the absorption edge of the characteristic radiation concerned. Then we assume that the slight energy difference of the photons mainly associated with I , permits approximating it to a monochromatic radiation for which the absorption coefficient, p ’ , of the pellet is considered constant at all levels. However, this assumption holds only for pellets made sufficiently “transparent” to I,: This is the case for pellets prepared as described below. The primary beam is considered parallel and penetrates the pellet with a n angle of incidence, CY. At depth h of the pellet, I , is reduced to II = I , exp( -1’ph cosec a) (1) The secondary intensity fraction emitted by the volume fraction, j % , dispersed in layer Adh and directed a t angle p toward the monochromator is after absorption through depth h
