Mixtures of lithium and sodium perchlorate could not be differentiated. The method can be dsed, however, to determine total lithium plus sodium. A mixture of lithium salts (lithium perchlorate-lithium benzoate) can also be differentiated as shown in Figure 4. The titration behavior of a mixture of lithium perchlorate and benzoic acid is unusual. When the benzoic acidlithium perchlorate mole ratio is less than or greater than one, the titration curve has three inflevtions (see Figure 4). .4n equimolar mixture of lithium perchlorate and benzic acid gives but two inflections. The first inflection in all three cases probably represents completion of the reaction
+
IAiC104 C‘6HjCOOH f Bu8Et?JOH C&COOLi BuaEtSCIOl HzO (2) -.f
+
+
When the benzoic xid-lithium perchlorate mole ratio is greater than one, the second inflection i!i due to the excess benzoic acid; when the ratio is less than one, the second inflection represents the titration of the excess lithium perchlorate. The third inflection (or the sccond inflection in the case of the equimolar mixture) represents the titration of the lithium benzoate. The titration curvw obtained for a mixture of sodium perchlorate and benzoic acid using two different indicating electrodes are shown in Figure 5. The volume of titrant to the first inflection is equivalent t o the amount of sodium perchlorate present and probably r e p r e s e h completion of the reaction.
1,000
t
900
8001
7001 6
I
4
4500 lL%
a -300 300
-200
2 00
- 100 Ordinate for Curw I 1
Ordinoto for Curve2
2 3 4 5 A. O.IN BU3EtNOH
6
7
Figure 5. Titration of sodium perchlorate-benzoic acid mixtures in pyridine Curve 1 ,
0.1 5 mmole sodium perchlorate and 0.50 mmole benzoic acid; glass electrode
Curve 2.
0.17 mmole sodium perchlorate and 0.50 mmole benzoic acid; mercuryfilm electrode
+
NaC104 f C&COOH BurEtXOH + CeHjCOOXa Bu3EthTC104 HLI (3)
+
+
The total volume of titrant to the second inflection is equivalent to the amount of benzoic acid present. The titration curve obtained with the mercury-film electrode is well defined with no irregularities. The relatively sharp second inflection in which the e.m.f.
approaches the limit for this particular electrode-solvent-titrant combination indicates that the sodium benzoate is nonacidic. Irregularities appear in the titration curve obtained with the glass electrode. The e.m.f. drop after the first inflection and the unsymmetrical nature of the second inflection could be explained by sudden changes in the sodium ion permeability of the glass electrode. The irregularities resemble those reported by Harlow ( 5 ) for the titration of phenol in pyridine with a quaternary ammonium hydroxide containing small amounts of sodium ion. Jt7e have found that the irregularities arising from these small amounts of sodium ion in the titrant or the sample can be eliniinated by the addition of small amounts of water to the solvent. This 15-ork ~t-illbe reported in the near future. LITERATURE CITED
(11 Banick, W. M., Jr., AKAL.CHEJI.34, 296 (1962). (2) Brummet, B. D., Hollweg, R. M., Ibid., 28,448 (1956). (3) Cundiff, R. H., Markunas, P. C., Ibid., p. 792. (4) Cundiff, R. H., Markunas, P. C., Anal. C h i n . Acta 21, 68 (1959). ( 5 ) Harlow, G. b.,~ A L CHEW . 34, 148 (1962). (6) Kolling, 0 . IT., J . C h e m Ed. 34, 170 (1957). ( 7 ) %IOrOS, s. A,, A N A L . CHEM. 34, 1584 (1962). (8) Seidell, A,, “Solubilities of Inorganic and Xetal Organic Compounds,” 3rd ed., p. 777, Van Nostrand, New York, 1940.
RECEIVED for review February 15, 1063. Accepted June 25, 1903.
Direct Analysis of Oxygen-1 8 in Organic Compounds C. GARDNER SWAIN, GEN-ICHI TSUCHIHASHI, and LYNN J. TAYLOR Department of Chemistry and laboratory for Nuclear Science, Massachusetts Institute o f Technology, Cambridge, Mass.
b By repeated scanning of the molecular-weight regions of inass spectra and careful measurement of peak intensities, it is possible to determine the oxygen18 content of many organic compounds without the need for chemical conversions and with a precision sufficient for most tracer and isotope-effect experiments, provided the iracer enrichment is about 5%.
A
LTHOUGH SEVERAL methods
have been suggested for analysis of isotopic composition of the oxygen in organic c~ompountts, most involve the construction of relatively complex apparatus and none appears to be general :iiid completely satidfactory (19). Doer-
ing and Dorfman (6) adapted the Unterzaucher procedure for quantitative oxygen analysis (21, I , 10) to the determination of oxygen-18 content and reported good results with a variety of compounds. However, more recent work (2, 3 ) indicates that the accuracy of the method is limited by the need to apply a correction for the reaction of hot carbon with silica. Anbar, Dostrovsky, and coworkers ( 2 ) reported that they have made many attempts to develop a general method for isotopic oxygen analysis, but without success. New procedures have been suggested and applied in particular cases (1.3, 5 . 7 , Q), but their general applicability has yet to be demonstrated.
In view of thit hituntion, n e have examined the possibility of determining oxygen-18 content by direct massspectrometric analysib. This technique has already been applied in case? where it was desired to dcterrniiie the isotopic composition of a particular oxygen atom (11, 2 2 ) . Such a method would avoid most of the difficulties of chemical conversion processes, such as incomplete conversion and dilution effects. Contamination by oxygen-containing impurities such as witer, carbon dioside, or oxygen should bt. less serious if a method is ba.ed on direct analysis than if chemical conversion is employed (8). We have developed a procedure which appetlrs to be suitable for :tnalysis VOL. 35, NO. IO, SEPTEMBER 1963
0
1415
Table 1.
Typical Analyses
Compound Benzyl alcohol Benzvl alcohol .~~~ .. . ~ ~ Phenh Phenol Benzyl phenyl ether Benzyl phenyl ether ~
0lS/O 16 0.06288 =I= 0.00016 0 .~ 0 6 2 7.l A 0.00008 ~
6.04230 =I= 0,00004 0.04227 i 0.00004 0.04351 & 0.00002 0.04349 =I= 0.00002
of the oxygen-18 content of many organic compounds. A similar technique has been applied for analysis of sulfur isotopes in methyl sulfide (14, 16). The sample is introduced directly into a recording mass spectrometer and the molecular-weight region scanned repeatedly. Although automatic switching between peaks was not employed in this study, switching circuits have been used in conjunction with a singlecollector mass spectrometer (16, 18). The 0 1 8 / 0 1 6 ratio is then obtained from the mean intensity ratio of the peak which is two units above the molecular 2 peak) to the molecular weight (M weight peak ( M peak). Because of the high natural abundance of carbon-13, it is usually necessary to subtract from the 2 ) / X ratio the contriobserved (M butions arising from molecules containing two atoms of carbon-13 or one of carbon-13 and one of deuterium or oxygen-17; these contributions may be calculated from the formula of Beynon (4). Uncertainties in the contributions become insignificant if a sufficient enrichment (usually 3y0 or more) of oxygen-18 is employed. For very precise work one can check the carbon13 content of each sample by detcrmining the (A4 l)/M ratio. This method has been applied to samples of benzyl alcohol, phenol, and benzyl phenyl ether containing 3 to 6% of 018. Typical results are shown in Table I. Errors are standard deviations, calculated from the results of repeated scans of the same sample. Sample-tosample reproducibility is demonstrated by the duplicate determinations on the same material. The accuracy of the method is not necessarily as good as its reproducibility. With a recording mass spectrometer the accuracy of an analysis can be no better than the accuracy of galvanometer calibrations. Isotope effects (reproducible but not accurately known) may cause small errors if the compound fragments extensively in the mass spectrometer. The procedure of sample introduction may give rise to slight isotopic fractionation. However, it is usually possible to design experiments so that one need know only the isotopic content relative to that of a standard sample-e.g., reactant, or product after complete reaction-of the same com-
+
+
+
1416
0
ANALYTICAL CHEMISTRY
pound, which may be analyzed under the same conditions. This technique is obviously limited to compounds which are sufficiently volatile for mass-spectrometric analysis and which have substantial molecular weight peaks (or peaks from oxygencontaining fragments) in their mass spectra. In certain cases--e.g., compounds containing deuterium, sulfur, or chlorine-the presence of a heavy isotope of another element will make the analysis more difficult. For compounds showing much fragmentation, fragmentation may be decreased by use of a lower than usual ionization potential closer to the appearance potential of the parent ion. Memory effects resulting from contamination of the inlet system with previously-analyzed samples were not important in this work. A standard sample was analyzed before and after the enriched sample, with excellent agreement. Table I shows the reproducibility of analyses of enriched samples. Of course, memory effects may vary depending on the nature of the sample, temperature of the inlet system, the efficiency of the pumping system, and the time between analyses. In some cases it might be desirable to flush the inlet system with one or two portions of the sample, or of a compound of lower molecular weight, before performing the analysis. Ion-molecule reactions, which can lead to ions which contain an extra hydrogen atom, were not important in this work. The intensity ratios were not significantly pressure-dependent, as they would have to be if bimolecular collisions were involved. They were also unaffected by electron energy (from 12 to 20 e.v. for benzyl alcohol). The C13/C12 ratios obtained from the data were in the natural-abundance range. If cases were encountered where ion-molecule reactions were serious, one could correct for them by 2 ) / M ratios meassubtracting ( M ured on samples of enriched and normal compound a t the same pressure.
was reduced to benzyl alcohol-018 with lithium aluminum hydride (1'7). All materials were purified by gas chromatography. Method of Analysis. Mass spectrometric analyses were performed on a Consolidated Electrodynamics Model 21-103C recording mass spectrometer. The inlet system was maintained a t 150' C. The background of the mass region to be recorded was always checked before the sample introduction and the preceding sample pumped off until the highest peak was reduced to less than 0.07% of the M peak height subsequently measured. To ensure maximum sample-to-sample reproducibility, the various steps of the sample introduction and analysis procedure were performed according to a time schedule and different samples of each compound were analyzed a t the same pressure. The molecular-weight region of the spectrum was scanned 10 to 20 times. Peak intensities were measured by counting whole divisions on the record and interpolating between divisions with the aid of a Bausch and Lomb Measuring Magnifier (No. 8134-35). SAMPLE CALCULATIONS
The method of calculation may be illustrated by the analysis of a sample of phenol. Raw data obtained from 10 scans are listed in Table 11. Averaging the isotope ratios obtained from the 10 scans gives 0.06806 for (M+l)/X and 0.04422 for (M+2)/M. For phenol (CsHeO), the statistical formulas of Beynon (4) become
D 2 "x2 (g)+ 15 (m) C'3 -k 15 (a) =
M
+
EXPERIMENTAL
Materials. Phenol-018 was prepared by the fusion of sodium hydroxide-0'8 with sodium benzenesulfonate (12). Benzyl phenyl ether-018 was prepared from the phenol by the action of benzyldimethylsulfonium tosylate in basic aqueous solution (20). Benzyl alcohol-018 was prepared by a four-step method. Benzoic acid-018 was obtained by refluxing water-018 with benzotrichloride. The benzoic acid-018 was converted to benzoyl chloride-018 by the action of thionyl chloride. The reaction of benzoyl chloride-018 with the potassium salt of triphenylcarbinol afforded triphenylinethyl benzoate-carbonyl-0'8, which
Since the ratios D/H and 017/016 are small, they need not be known with great precision. Here we assume the natural-abundance ratio (0.00016) for D/H. Since the measured (M+l)/M ratio exceeds by 0.0011 that determined on a sample of ordinary phenol under the same conditions, we assume that Ol7/O16 exceeds natural abundance (0.0004) by that amount-Le., that 017/016 = 0.0011 0.0004 = 0.0015. We can now determine C13/C12 by substituting these values and the observed (M l ) / M ratio into Equation 1. The result is Cla/C12 = 0.01093. We now obtain 018/016 = 0.04227 by substituting C1d/C1*ID/H, O17/Oth, and the observed ( M + 2 ) / M ratio into Equation 2.
+
+
(M Table It.
Peak intensities (divisions on record) Scan Mass 94 ( M ) Mass 95 ( M 1) Mass 96 ( M
+
1 2 3 4 5 6 7 8 9 10
486.6 486.4 484.1 479.5 480.3 479.8 475.1 477.1 474.6 472.8
33.19 33.05 32.91 32.68 32.65 32.66 32.31 32.40 32.28 32.28
+ 2)
21.59 21.47 21.37 21.37 21.28 21.16 20.97 21.12 20.96 20.81
An alternative melhod may be illustrated by the determination of the oxygen-18 content of labeled benzyl alcohol. Here the prt:sence of a large M- 1 fragment complicates calculations by the above method. How2)/M ever, by subtracting ],he (A1 ratio measured on an unlabeled sample from that determined on the labeled sample, we can determine the excess 0l8/Ol6 ratio to a reasonable approximation. For example, the ( M 2)/M ratio of a sample of labeled benzyl alcohol was 0.06557, while that determined on a similarly prepared unlabeled sample was 0.00472. Therefore, the 018/016 ratio in excess of natural abundance was appro qimately 0.06557 - 0.00472 = 0.06055. The total 018/016 ratio (0.06285) was then obtained by adding the natural abundance ratio (0.00200). A more complicated calculation, in which corrections were applied for exces: oagen-17 content of the labeled sarrple and for contributions to the M , M 1, and A 1 2 peaks by fragments which contained heavy isotopes but had lost one hydrogen atom (as described below), resulted in a figure of 0.06271 for total
+
+
+ +
+
+
=
Thus A is 0.859 and the peak intensity ratios, corrected for fragmentation, are
+ 2)/M
0.04437 0.04414 0.04414 0.04457 0.04431 0.04410 0.04414 0.04427 0.04416 0.04401
The isotope ratios can be determined by substituting these intensity ratios into Equations 1 and 2 andproc eeding as described above. ACKNOWLEDGMENT
The authors acknowledge the helpful advice and cooperation of Klaus Biemann. LITERATURE CITED
+
where (M 2)obs is the observed peak intensity, (ill 2),,, is the intensity due to unfragmented molecules of mass 31 2, and A is the ratio (M - 1)/M which would be obtained in the absence of isotopes. Similarly,
(1) Aluise, V. A., et al., ANAL.CHEM.23, 530 (1961). (2) Anbar, M., Dostrovsky, I., Klein, F., Samuel, D., J . Chem. SOC.1955, 155. (3) Bender, M. L., Kemp, K. C., J . Am. Chem. Sac. 79, 117 (1957). (4) Beynon, J. H., "Mass Spectrometry
The fragmentation ratio A is now determined by successive approximation. Initially, we assume that A = 80.50/ 100 = 0.8050. Equations 3 through 5 then become
1960. (5) Dahn, H., Moll, H., MenassB, R., Helv. Chim. Acta 42, 1225 (1959). (6) Doering, W. von E., Dorfman, E., J . Am. Chem. SOC.75, 5595 (1953). (7) Lauder, I., Wilson, I. R., AustralianJ. Chem. 12, 613 (1959). (8) Ibid., 14, 166 (1961). (9) Lauder, I., Zerner, B., Ibid., 12, 621 (1959). (10) Lee, T. S., Meyer, R., Anal. Chim. Acta 13, 340 (1955). (11) Long, F. A., Friedman, L., ,T. Am. 72,3692 3692 (1950). Chem. SOC.72, Makolkin. . I., Acta Physicochim. (12) Makolkin, Phusicochim. U.R.S.S. 16.' 16, 88 (1942). " (13) Rittenberg, D., Ponticorvo, L., Intern. J . Appl. Radiatim and Isotopes 1, 208 (1956). (14) Saunders, W. H., Jr., Asperger, S., J . Am. Chem. SOC.79, 1612 (1957). (15) Schutten, J., Boerboom, A. J. H.,
+
+
+ 2)m = 0.472 - (0.805)(0.031) (At +
and its Applications to Organic Chemistry," p. 296, Elsevier, Amsterdam,
'
(3f
1)cor =
7.720
+
(M
suming that isotopes have a negligible effect on the estent of fragmentation, we can write
01*/016.
A more rigorous altmnative method involves a successive approximation procedure to correct fcr fragmentation of one hydrogen atom, as the following analysis of a sample of normal (unlabeled) benzyl alcohol demonstrates. A similar analysis was riade on data for an Ol8-enriched samde. For the normal sample, the peak intensities relative to 100 for the M oeak were 0.031 3, 0.472 for Id 2, 7.72 for for 111 M 1, and 80.50 for M - 1. The hl 4 and ill - 2 peaks \$ere both very small. Except for the M - 1 peak, there is a contribution to each peak arising from a fragment containing one or more heavy isotopes. For example, molecules containing one atom of carbon-13 should all appear a t J1 1, but some will fragment and appear at mass Ai' instead; consequently we must correct for the contribution of tlicie fragmerits to the d l peak. ils-
+ 1)/M
0.06821 0.06795 0.06798 0.06815 0.06798 0.06807 0.06801 0.06791 0.06802 0.06827
+
+
(M
- 1)oba = 80.50
(0.859)(93.70) = 80.49
Peak Intensities in Analysis of Phenol Sample
(Mho? = 100.00
- (0.805)(0.447)
- (0.805)(7.360) =
=
0.447
= 7.360
Hauw, T. v. d., Monterie, F., Appl. Sci.
94.08
Res. Sect. B 6, 388 (1957). (16) Swain, C. G., Thornton, E. R., J . Org. Chem. 26, 4808 (1961). (17) Swain, C. G., Tsuchihashi, G., Massachusetts Institute of Technology,
But Equation 6 is then (M
-
l)o~ = s80.50 = (0.805)(94.08) = 75.73
Therefore the true value of A must be larger than 0.805. As a second ap80.50 proximation, we take -4 = 0.805 X = 0.856. Repetition of the above procedure (using 0.856 instead of 0.805) gives
Cambridge, Mass., unpublished work,
7m3
(M
-
1)oba = 80.50 (0.856)(93.72) = 80.22
The agreement is now much better, hut A is still too low. We take A = 0.856 (80.50)/80.22 = 0.8590 and rep w t the steps once more to obtain
1962. (18) Taylor, C., Arkiv fur Fysik 8 , 201 (1954). (19) Taylor, L. J., Ph.D. thesis in organic chemistry, pp. 9-13, Massachusetts Institute of Technology, Cambridge, Mass., January 1963. (20) Ibid., pp. 63-9. (21) Unterzaucher, J., Analyst 77, 584 (1952). (22) Wiberg, K. B., J . Am. Chem. SOC.75, 2665 (1953).
RECEIVED for review March 6, 1963. Accepted June 12, 1963. Supported in part by the Atomic Energy Commission under Contract no. AT(30-1)-905 and by N. S. F. and N. 1. H. predoctoral fellowships to 1,. J. rr.
VOL 35, NO. 10, SEPTEMBER 1963
e
1417
The Differentiation of the n-AlkanaI 2,4 -Dinitrophenylhydrazones by Infra red Spectrometry HARRY G. LENT0 and JAMES A. FORD Campbell Soup Co., 7 00 Markef Sf., Camden 7, N . 1.
b A method is described for the differentiation of members of the homologous series of saturated aliphatic aldehydes b y means of the infrared spectra of their 2,4-dinitrophenylhydrazones. In this method, the chain length in the parent carbonyl compound is determined from a demonstrated linear relationship between the number of methylene groups present and the ratio of the absorbance of the CH2 and NH bands. The nalkanal 2,4-dinitrophenylhydrazones are prepared as a potassium bromide disk and the absorbance ratio of the CH2 and NH band is calculated from measured absorbances. The chain length of the parent carbonyl i s then determined by a calibration curve prepared from a series of known n-alkanal derivatives, The method is applicable to the differentiation of the n-alkanals from CB to C16. Exact duplication of experimental conditions is not required and the results are independent of concentration of sample in the KBr.
from the hydrogen stretching and bending modes of the CH2 group increases relative t o the band intensity of the 2,4-DNPH portion of the molecule. These investigators made use of this characteristic behavior to dktinguish the 2-alkenalb from the 2,4-dienals and to differentiate individual members within each of these groups. A detailed study of the manner in which infrared spectrometry has been used to differentiate individual members of a homologous series of n-alkanal 2,4DXPH’s is presented in this paper. The method, which is similar t o that employed by Stitt et al. (IO), is based on the linear relationship between the ratio of the absorbance of the CH2 to ?;H stretching modes and the chain length of the parent carbonyl. This ratio permits the number of methylene groups in the molecule to be calculated. and from this, the total number of carbon atoms in the n-alkanals can be determined. EXPERIMENTAL
All reagents used in this study were c.P., or equivalent grade. Potassium bromide was infrared quality, 200 mesh. This reagent was stored in a desiccator. Apparatus. The Wig-L-Bug was a n automatic, mechanical grinder from Spex Industries, Inc., Scotch Plains, N. J. The hydraulic press was Wabash Model 12-10s from Wabash Metal Products, Wabash, Ind. The infrared sDectrouhotometer was a Model 221 from ihe PerkinElmer Corp., Xorwalk, Conn. Preparation of 2,4-DNPH’s. The 2,4-dinitrophenylhydrazine derivatives Reagents.
I
that carbonyl compounds play an important role in the flavor of many foods. To elucidate the nature of these compounds, the aldehydes and ketones are usually isolated as their 2,4-dinitrophenylhydrazones (2,4-DXPH), since these derivatives are easily purified t o yield crystalline compounds. Among the unique features of the 2,4DXPH’s are their characteristic color in neutral and in basic solution. The ultraviolet and infrared absorption spectra of these compounds are also quite useful for identification. For example, Braude (4), Corbin, Schwartz, and Keeney ( 5 ) , and Timmons (11) have employed ultraviolet absorption q~ectrometryt o determine the aliphatic, aromatic, or olefinic nature of the parent carbonyl. Similarly, in a detailed spectrophotometric study of various 2,4-DKPH’s, Jones ( 7 ) has reported that the aliphatic, aromatic, and olefinic derivatives could be characterized from their infrared spectra, although individual members within each group could not be distinguished by this technique. Recently, Stitt et al. (IO) have shown that the intensity of the bands arising T IS WELL RCCOGKIZCD
1418
ANALYTICAL CHEMISTRY
Table 1.
Alkanal Propanal Butanal Pentanal Hexanal Hept anal Octanal ?A-
Eonanal
of the Ca through the C1, and CL6 alkanals were prepared by reacting alcoholic solutions of each aldehyde with a n alcoholic-sulfuric acid solution of 2,4-dinitrophenylhydrazine according to the procedure described by Shriner and Fuson (9). The derivatives were recrystallized from ethanol at lrast three times or until the melting point was constant. The criterion of purity mas established through analysis of their nitrogen content according to the semimicro Kjeldahl procedure described by Allen ( I ) . Table I shows the results of these analyses. Stock solutions of each alkanal 2,4DXPH were prepared by dissolving 5 mg. of each derivative in 10 ml. of chloroform. Infrared Spectrophotometry. To 150 mg. of potassium bromide contained in a 5-mL beaker was added a n aliquot of the stock solution. The solvent was removed and the sample evaporated to dryness under a n infrared heater to precipitate the 2,4D N P H onto the potassium bromide. The sample mas then transferred t o a stainless steel vial of the Wig-L-Bug. Although not quantitative, the transfer mas done as completely as possible to ensure that a nearly theoretical amount of derivative would be contained in the finished pellet. To effect homogeneity, the sample-KBr mixture was ground for exactly 7 seconds in the Wig-L-Bug. Grinding times longer than 7 seconds apparently reduced the KBr particle size, which led to greater light scattering a t lower wavelengths. This tended to obscure the region containing the bands of interest, Following homogenization of the sample, the contents were transferred, as quantitatively as possible, to the pellet die by gently tapping the sides
n-Alkanal 2,4-Dinitrophenylhydrazones--Melting Analyses, and CH2/NH Absorbance Ratio
Melting point, “C. Detd. Reptd. 156 123 106 110 109 108 108 108 106
155 ( 1 ) 120 (6) 106 ( 9 ) 110 ( 8 ) 108 ( 8 ) 108 (1) 109 (1) 104 ( 1 ) 104 ( 1 )
Sitrogen, yo Calcd. Detd. 23.46 22.19 21.09 19.97 18.90 18.13 17.30 16.30 15.72 13.30
Decanal Gndecanal Hexadecanal a Average of five determinat,ions f l std. dev.
23.46 22.21 21.04 19.99 19.03 18.17 17.38 16.65 15.99 13.32
Point, Nitrogen
CH,/SH ratio“ 0.29 i 0.004 0.53 f 0.011 0.76 =k 0.017 0.93 rt 0.027 1 . 1 3 0.024 1.36 =k 0.029 1 . 6 3 i 0.027 1.85 f 0.042 2.09 0.051 3.29 3= 0.098
* *
