It was necessary to extract mercury as the dithizonate into chloroform and to prepare the electrode from this solution. Attempts to evaporate directly the aqueous (0.1N HCl) absorbing solution following addition of the graphite powder resulted in complete loss of mercury by vaporization. Silver was chosen as the internal standard because it forms the dithizonate under the same conditions as mercury and also would be expected to be essentially absent in apple tissue. Formation of silver dithizonate is somewhat slower than that of mercury dithizonate. It was essential to extract the aqueous absorbing solution with dithizone in chloroform for no less than 3 minutes. Shorter equilibration times resulted in a considerable reduction in the quantity of silver extracted. To assure that silver was efficiently extracted, spark source mass spectrometric determinations were made on silver extracted under conditions specified when silver was present in the aqueous phase at several concentrations. Copper was used as the internal standard, because it also rapidly forms the dithizonate under the conditions that mercury does. Over 90% of the silver was consistently extracted during 3 minutes of equilibration. Partitioning for periods up to 10 minutes did not markedly increase extraction efficiency. The method as described is sensitive to about 2 ppb of mercury in apples. Depending on the level of mercury found in
control fruit, the sensitivity could be increased by extraction of a larger volume of the absorbing solution. In several tests, the entire solution (50 ml) was extracted with proportionately larger volumes of each reagent and the electrode prepared to contain 5 times the amount of equivalent sample. The resulting spectrograms were free of interfering lines and permitted quantitative densitometric measurements. The advantages of spark source mass spectrometry include versatility, sensitivity, determination of more than one element at the same time, positive identification based on analysis of several isotopes of a desired element, and possible obviation of interference by choice of the proper isotope(s) for measurement. It is predictable that this technique will be applied increasingly to future determination of trace metals in biological investigations. ACKNOWLEDGMENT
The authors thank Michael Szkolnik for providing samples of field-treated apples and Richard Hicks for his technical assistance. RECEIVED for review June 30, 1969. Accepted August, 25, 1969. The support of Corning Glass Works for this investigation is appreciated.
Rapid Micromethod for Location of Ene-yne and a=Hydroxy Conjugated Diene Systems in Straight-Chain Compounds G . F. Spencer, R. Kleiman, F. R. Earle, and I. A. Wolff Northern Regional Research Laboratory, Peoria, III. 61604 FORUNSATURATED aliphatic compounds with known functional groups, oxidative cleavage at the sites of unsaturation and identification of the fragments formed constitute a convenient means of bond location. Rapid analytical procedures coupling ozonolysis and gas chromatography have been developed (1-3). Such methods have generally been restricted to locating double bonds. Triple bonds have been located by ozonolysis in acidic media (4, 5), but recovery and identification of the acids produced are time consuming. The procedure described here is rapid, facile, specific for double and triple bonds, and is applicable to very small samples. The method has a further advantage: It can be used to locate both the bonds and the hydroxyl group in ahydroxy conjugated dienes. EXPERIMENTAL
Materials. Acetylenic hydrocarbons and methyl oleate were obtained in high purity from commercial suppliers. Methyl stearolate (9-octadecynoate) was synthesized (6). Methyl tarirate (6-octadecynoate) was isolated by preparative thin-layer chromatography (TLC) on AgN0,-impregnated
plates (7) from esters derived from Picramnia sellowii seed oil (8). Methyl crepenynate (cis-9-octadecen-12-ynoate) (9) was isolated by preparative gas-liquid chromatography (GLC) followed by preparative TLC (7) of esters from Crepis a/pina seed oil. Methyl 17-0ctadecen-9-ynoate, methyl dimorphecolate (9-hydroxy-trans-l0,trans-l2-octadecadienoate),and methyl coriolate (13-hydroxy-cis-9-trans-11-0ctadecadienoate) were available at this laboratory (10-12). Ozone was generated as described by Bonner (13). Apparatus and Procedure. A 0.2x solution of sample was prepared in ACS grade methanol in a conical, 12-1111 centrifuge tube. An ozone-oxygen mixture was bubbled through the solution at approximately 10 ml/minute at room temperature. Usually the solution was ozonized 1 minute/mg of sample. Two 15-pl portions of the ozonized solution were injected directly into a Packard 7401 gas chromatograph equipped with dual columns and independent flame-ionization detectors. Two glass columns were used: one 12 ft X inch
(7) R. Kleiman, G. F. Spencer, F. R. Earle, and I. A. Wolff, Chem. Ind. (London), 1967, 1326. (8) G. F. Spencer, R. Kleiman, F. R. Earle, and I. A.Wolff, Lipids,
in press. (1) 0.S. Privett and E. C. Nickell, J . Amer. Oil Chem. SOC.,39, 41 4 (1962). (2) R. A. Stein and N. Nicolaides, J. Lipid Res., 3, 476 (1962). (3) V. L.Davison and H. J. Dutton, ANAL.CHEM., 38,1302 (1966). (4) C. D. Hurd and R, E. Christ, J . Org. Chem., 1, 141 (1936). (5) F.Bohlmann and H. Sinn, Chem. Ber., 88,1869 (1955). (6) R. 0.Butterfield and H. J. Dutton, J . Amer. Oil Chem. SOC., 45, 635 (1968). 1874
(9) K. L. Mikolajczak, C. R. Smith, Jr., M. 0. Bagby, and I. A. Wolff, J . Org. Chem., 29,318 (1964). (10) R.G.Powell and C. R. Smith, Jr., Biochemistry, 5,625 (1966). (11) C.R. Smith, Jr., T. L. Wilson, E. H. Melvin, and I. A. Wolff, J. Amer. Chem. SOC.,82,1417 (1960). (12) W. H. Tallent, J. Harris, and I. A. Wolff, Tetrahedron Lett., 36, 4329 (1966). (13) W. A. Bonner, J. Chem. Educ., 30,452 (1953).
ANALYTICAL CHEMISTRY, VOL. 41, NO. 13, NOVEMBER 1969
packed with 5 LAC-2-R 446 on Chromosorb W AW-DMCS and one 4 ft X 'I4 inch packed with 5 Apiezon L on Chromosorb W AW-DMCS. The temperature of the inlet was 200 "C and the column oven was programmed from 40" to 200 "C at 5 "C/minute. Sometimes a 5-minute hold at the starting temperature improved the resolution of the more volatile components. Components were identified by their equivalent chain lengths (ECL, based on saturated methyl ester standards) (14-16), from both columns. After peak areas were measured with an Infotronics electronic integrator, area percentages of the components were calculated. Compounds containing both double and triple bonds were analyzed by this procedure and, again, by low-temperature ozonolysis in dichloromethane followed by reduction with triphenylphosphine (2, 16). To determine optimum reaction time, one 5-mg sample of 5-decyne dissolved in 2.5 ml of methanol was ozonized for seven 1-minute intervals. After each interval, samples were taken and analyzed by GLC. RESULTS AND DISCUSSION
Monoenes and Monoynes. Diaper and Mitchell (17) established that the major product from the ozonization of ethyl 10-undecenoate in ethanol followed by thermal decomposition of the ozonolysis product is diethyl sebacate. Under our conditions, although direct GLC of an ozonized methanolic solution of methyl oleate showed significant amounts of methyl esters as cleavage fragments, the major products were aldehydes. Ozonization of compounds containing triple bonds yielded primarily the ester products, along with small amounts of free acids, but no aldehydes. Presumably, these products arise from thermal decomposition of ozonides and/or other ozonolysis products (18) during chromatography. Under our procedures of ozonolysis and GLC, olefinic and acetylenic bonds were cleaved to two different sets of products as follows (Scheme 1):
17 mole %. Methyl butyrate and methyl caproate were the major products from ozonization-GLC analysis of 4-decyne; whereas from 1-decyne, methyl pelargonate was the only ester detected. Methyl formate evidently did not separate from the methanol. Methyl stearolate yielded equal amounts of methyl pelargonate and dimethyl azelate as calculated from peak areas. Ozonization-GLC of methyl tarirate gave methyl laurate and dimethyl adipate in an approximate ratio of 2 :1. In relation to the chain lengths of the fragments, these area ratios are in good agreement with those previously found for other ozonolysis products (16). Compounds Containing Ene-yne Systems. Ozonolysis of methyl crepenynate at low temperature in CHzClt (16) did not appear to cleave the triple bond. The two fragments formed were methyl azelaaldehydate and a component with ECL's (7.9 on Apiezon L and 10.0 on LAC-2-R 446) consistent with those of a 9-carbon aldehyde with one triple bond (14,16). The only detectable fragment from the low-temperature ozonization of methyl 17-octadecen-9-ynoate had ECL's predicted for a 17-carbon aldehyde ester with one triple bond. When other samples of the two esters were ozonized in methanol at room temperature, GLC gave the expected ester and acid products from the triple bonds and the aldehyde and ester products from the double bonds. The products from methyl crepenynate and their area percentages are shown below (Scheme 2).
CH,-[CH21,-C< [7.6%]
t
t
t
t
-
t
.
t
The products from ozonization of methyl oleate in methanol at room temperature were: pelargonaldehyde, 41 %; methyl azelaaldehydate, 33 %; methyl pelargonate, 8 %; dimethyl azelate, 11%; and seven other unidentified components totaling only 7 %. After 4 minutes' ozonization of 5-decyne, GLC peak area percentages were: methyl valerate, 66%; Sdecyne, 16%; and valeric acid, 18%. Correcting for flame ionization response of oxygenated carbons (16) and converting to mole per cent gives : methyl valerate, 69 ; 5-decyne, 9 %; and valeric acid, 22%. After 7 minutes' ozonization the 5-decyne had completely reacted and the composition of the mixture was: methyl valerate, 83 mole % and valeric acid,
(14) T.K. Miwa, J . Amer. Oil Chem. Soc., 40,309 (1963). (15) T. K. Miwa, K. L. Mikolajczak, F. R. Earle, and I. A. Wolff, ANAL.CHEM.,32, 1739 (1960). (16) R. Kleiman, G. F. Spencer, F. R. Earle, and I. A. Wolf€, Lipids, 4, 135 (1969). (17) D. G.M. Diaper and D. L. Mitchell, Can. J . Chem., 43, 319 (1 965). (18) E. H.Pryde, D. E. Anders, H. M. Teeter, and J. C. Cowan, J . Org. Chem., 25, 618 (1960).
/O
0-H
Two other peaks, tentatively identified (ECL from LAC-2-R 446 column only) as dimethyl malonate and 3-nonynal, made up 7 x of the total peak areas. The acetylenic aldehyde would result from incomplete ozonization of the parent ester. The minor amounts of difunctional compounds containing free acid groups formed from the central portion of the ester were not identified. The methyl caproate and methyl azelaaldehydate delineate the structure of the parent compound. Low-temperature ozonolysis (16) confirms the position of the double bond and would be helpful in resolving isomeric mixtures. Hydroxy Conjugated Dienes. Fatty acids with conjugated unsaturation and a hydroxyl group CY to one double bond have been found in several seed oils (11, 12, 19). Although structural evidence for such acids was obtained through chemical and spectrometric means, location of the functional groups involved many steps (11, 12). Room temperature ozonization of methyl dimorphecolate and methyl coriolate in methanol, followed by GLC, produced a unique set of fragments from each ester. These products and their corresponding GLC area percentages are illustrated in the following scheme (Scheme 3).
(19) F. D. Gunstone, "An Introduction to the Chemistry and Biochemistry of Fatty Acids and Their Glycerides," 2nd ed., Chapman and Hall Ltd., London, 1967,p 23.
ANALYTICAL CHEMISTRY, VOL. 41, NO. 13, NOVEMBER 1969
e
1875
was caproic acid (identified after esterification with CH2N2). The splitting-out reaction was not complete, however, because a-hydroxy heptanal was also formed. Since neither the acids nor the a-hydroxy fragments were observed following roomtemperature ozonolysis in methanol, this procedure is more effective and convenient. It yields fragments which are easily identifiable without supplementary reactions.
+ CH,-JCHiJrC”o
\o-cnI
(4.6%)
CHi-(CHi),-CH-C=C-C=C-(CH~),-C= nt I OH
,
ACKNOWLEDGMENT
/o
n n n
O-CHl ICoriolatr)
The geometry of the double bonds apparently had no effect on the cleavage pattern. Splitting-off of the carbon atom adjacent to the hydroxylated atom was observed upon ozonolysis in CHzClz at dry-ice temperature (20); the fragment formed from methyl coriolate (20) W. H. Tallent, J. Harris, G. F. Spencer, and I. A. Wolff, Lipids, 3, 425 (1968).
The authors thank R. 0. Buttefield for methyl stearolate, R. G. Powell for methyl 17-octadecen-9-ynoate, W. H. Tallent for methyl coriolate and methyl dimorphecolate, and T. K. Miwa for stimulating suggestions and discussions.
RECEIVED for review June 30, 1969. Accepted September 5, 1969. Presented at the Great Lakes Regional Meeting of the American Chemical Society, DeKalb, Ill., June 5-6, 1969. The Northern Regional Research Laboratory is headquarters for the Northern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. Mention of trade or company names is for identification only and does not imply endorsement by the Department.
Indicator Titrations in Tetramethylurea Siegmond L. Culp and Joseph A. Carusol Department of Chemistry, Uniaersity of Cincinnati, Cincinnati, Ohio 45221 RECENTLYwe reported on tetramethylurea (TMU) as a solvent for potentiometric acid-base titrations ( I ) . It was shown that TMU is indeed a useful solvent and is suitable for differentiating a wide range of acids and bases. Results of the first study indicated that perhaps other methods of detecting the end points also might be investigated. Indicators have proved to be especially helpful in the detection of end points in cases where especially steep potential curves are observed in nonaqueous titrations ( 2 ) . Because the titration curves obtained in TMU tended to be rather steep, the investigation of suitable indicators seemed an appropriate study. The most commonly used indicators in nonaqueous titrimetry are thymol blue (thymolsulfophthalein), azo violet [4-(pnitropheny1azo)-resorcinol], and crystal violet (hexamethyl-prosaniline hydrochloride) (3). Crystal violet, however, is used almost exclusively in acetic acid while the first two mentioned have been found to be operative in a wide variety of solvents ( 4 ) . Fritz has recommended thymol blue as the most generally satisfactory indicator in DMF while azo violet, with a more basic transition point, was found to be preferable when very week acids were titrated (5). 1
To whom all communications should be addressed.
(1) Siegmond L. Culp and Joseph A. Caruso, ANAL.CHEM., 41, 1329 (1969). (2) I. Gyenes, “Titration in Non-Aqueous Media,” D. Van
Nostrand Co., Princeton, New Jersey, 1967, p 198. (3) Zbid.,p 204.
(4) J. T. Stock and W. C. Purdy, Chemist Analyst, 48,50 (1959). ( 5 ) J. S . Fritz, ANAL.CHEM., 24, 306 (1952).
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EXPERIMENTAL
All of the indicators used were obtained from Eastman and were “white label” or reagent grade. These were dissolved in a sufficient amount of purified tetramethylurea to produce a 0.3% solution. From two to five drops of indicator solution were used in titrating samples of benzoic acid, phenol, or 1,3-diphenylguanidine. All indicator titrations were monitored potentiometrically to establish the color change which best corresponded to the equivalence point. The purification of other reagents, descriptions of apparatus, and experimental procedures, have been discussed previously ( I ) . RESULTS AND DISCUSSION
The indicators evaluated for the titration of acids were thymol blue, phenolphthalein, azo violet, alizarin yellow, and curcumin. Thymol blue, phenolphthalein, and azo violet all performed satisfactorily in the titration of benzoic acid. With alizarin yellow, the color change did not occur until well after the benzoic acid equivalence point had been passed. Curcumin was evaluated only in the titration of phenol. Azo violet and curcumin were the only indicators with sufficiently basic transition points to produce a color change during the titration of phenol. However, the color transitions were not sufficiently sharp to permit satisfactory visual determination of the equivalence point. These indicators might be quite suitable, however, for the spectrophotometric titration of very weak acids in tetramethylurea.
ANALYTICAL CHEMISTRY, VOL. 41, NO. 13, NOVEMBER 1969
