lution. The EAE. for this maximum, calculated to be 1980 for a n alcohol solution, was 1670 for the aqueous solution. The p K value found for vanillin is 7 . 3 . Hence. it has a transition interval in a p H range slightly lower than that of phenolphthalein. It resembles the latter in being essentially a onecolor t!-pe indicator, as the acid form has almost a negligible absorption a t X 347 mp, where the basic form is a t its maximum. Three well-defined isosbestic points are evident at wave lengths 238, 259, and 316 mp. T h e fact that the absorbancies at the isosbestic points are not affected by p H changes offers some interesting possibilities in the examination of multicomponent systems. However, measurements a t these points require careful setting and checking of the wave length adjustment, as the absorption is markedly different a t wave lengths only slightly different from the proper values,
Table II. Stability of Vanillin Solution a t pH 10.75, Room Temperature
Date of Examination April 26, 1956 March 4, 1956 June 1, 1956 July 5, 1956 Oct. 15, 1956
Absorbance in 1-Cm. Cell 247 mp 347 mp 0.60 1.68 0.60 1.68 0.60 1.68 0.61 0.61
1.67 1.67
The observed’ differences in absorption may be concerned with a n equilibrium involving a change from a benzenoid to a resonance hybrid, to which the quinoid structure makes an important contribution, and analogous to the conduct of p-nitrophenol (6). H
O
0-
H
H
O
‘C’
0
nature and amount, use of larger TOTumes may cause trouble, and p H specification and check on its value is desirable. Other parahydroxyaldehydes may have dissociation constants of different magnitudes, and selection of specific p H conditions may be even more necessary for analysis of mixtures of these compounds. After this article had been prepared for publication, attention was called to a paper by Robinson and Kiang (9). in which the ionization constants of vanillin and two of itb isomers had been reported. The p K d u e reported for vanillin was 7.496.
The proportion of the two forms may be estimated for any pH value in the transition range from the equation pH
=
fill1 basic form i‘3 + log [full acid form
1
hssuming that one part of one form is detectable in the presence of 200 of the other, 9.6 and 5.0 represent the p H values a t the limits of the transition interval. Hence, p H values slightly greater than 9.6 and less than 5.0, respectively, are necessary for quantitative evaluations of the basic and acid forms, in order to secure theoretical conversion of 99.5%. values in excess of these figures may be advantageous to allow a margin of assurance for complete conversion. T h e use of a 10-ml. portion of the filtrate specified gives a p H of 11. If the extraneous materials in all genuine extracts are not of the same
LITERATURE CITED
(1) Englis, D. T., Hanahan, D. J., IND. ENG. CHEM., ANAL. ED. 16. 505 (1944).
(2) Englis, ’D. T., Manchester, Merle, ANAL.CHEIII.21, 591 (1949). (3) Englis, D. T., Wollermann, L. A . , Food Research 20, 567 (1955). (4) Ensminger, L. G., j.Assoc. O$c. Agr. Chemists 36. 679 (1953). Kolthoff, I. hi., Rosenblum, Charles, “Acid-Base Indicators,” p. 152, Macmillan, New York, 1937. Lemon, H. K., ANAL. CHEY.19, 84G ( 1947) * 17) Lemon. H. W,, J . Am. C h e w SOC.69, ’ 29O8’(1947): (8) Morton, R. A., Stubbs, A. L., J . Cheni. SOC.(London)1940, 1937. (9) Robinson, R. A., Kiang, A. K., Trans. Faraday SOC.51, 1398 (1955). RECEIVED for review March 31, t!M. .4ccepted February 7, 1957.
Analyses of a Chromatographic Fraction of Organic Extracts of Soils W. G. MElNSCHElN and G. S. KENNY Magnolia Pefroleom Co.,Field Research Laboratory, Dallas, Tex.
,An investigation of the chemical composition of a chromatographic fraction of the benzene-methanol extracts of soils i s described. The benzene eluates of the soil extracts from silica gel chromatographic columns contain high concentration of waxes of the types appearing in beeswax. Qualitative and semiquantitative analyses of the soil waxes are accornplished by use of chromatographic, infrared, and mass spectrometric methods in conjunction with hydrogenation studies. The types of acids and alcohols that form the wax esters are determined by converting the esters
to saturated hydrocarbons which are analyzed mass spectrometrically. The principal constituents of the waxes are normal aliphatic acids, normal primary aliphatic alcohols, and sterols. All the soil extracts contain pentacyclic and hexacyclic compounds which may b e triterpenes.
T
of the organic extracts of sediments are of interest because these extracts may be a source of petroleum, but the chemical nature of such extracts has not been extensively investigated because of their complexity. However, in recent years HE COMPOSITIOX
significant advances have been made in the analysis of organic mixtures. One of the more important factors contributing to these advances has been the modification of a commercial niass spectrometer to the analysis of high niolecular weight compounds by O’Seal and JJ7ier (6). The comprehensive review articles hy Dibeler (6) may be consulted for the accomplishments in the field of high mass spectrometry. This investigation of a chromatographic fraction of the organic extracts of soils illustrates how high mass spectrometric analyses may be used in conjunction with other study methods to deVOL. 2 9 , NO. 8 , AUGUST 1957
* 1153
termine the composition of complex lipides. The soil extract fractions are shown by chromatographic, infrared, hydrogenation, and mass spectrometric studies to contain high concentrations of waxes. The procedures employed to obtain information concerning the concentrations and molecular sizes of these xaxes and of the acids and alcohols that form these wax esters are presented. REAGENTS
Silica gel, commercial grade, Davison Co., 100 to 200 mesh, activated prior to use at 425' C. for 8 hours. Extraction and chromatographic solvents, commercial grade; distilled and checked t o contain less than 0.3 y per ml. of organic impurities which a r e not volatile under conditions eniployed for sample recovery. Carbon disulfide, reagent grade, J. T. Baker Co., distilled prior to use. Raney catalyst powder, activated b y the method of Pavlic and Adkins ( 7 ) . Urea, C.P. or equivalent, recrystallized from ethyl alcohol.
PROCEDURES
Sampling. The soil samples were obtained at a depth of approximately 3 feet so as t o minimize the inclusion of surface organic debris, roots, and vegetation. Extraction. Portions of 150 grains of each soil sample were extracted by lolling for 6 hours in a 500-ml., glass-stoppered, borosilicate glass bottle containing 25 0.5-inch diameter glass balls and 150 nil. of solvent, which was composed of 10 volumes of benzene and 1 volume of methanol. After this step, 10 nil. of distilled n a t e r was added t o each bottle t o assist in breaking t h e emulsions and the top 100 ml. of solvent was decanted through a glass wool filter to remove suspended particles. The extracts mere recovered from the solvents by blowing to constant nTeight a t 40" C. with air filtered through silica gel. All the extracts of each soil sample were combined. Silica Gel Chromatography. T h e soil extracts and their hydrogenated benzene eluates (desciibed below) \\ere fractionated on columns (17 to 19 om. in length and 9 mm. in diameter) containing 9 grams of silica gel. -4 30- to 50-1ng. portion of t h e sample, dissolved in 5-ml. aliquots of the solvents being used as eluents, was placed on t h e column prewet with 3 ml. of n-heptane, and the column was eluted successively with 15 nil. each of n-heptane, carbon tetrachloride, benzene, and methanol. The fractions eluted by each eluent were collected separately, and like fractions of the same sample mere combined. The fractions were recovered by blowing to constant weight at 40' C. Only the benzene eluates and samples derived
1 154
ANALYTICAL CHEMISTRY
from them were used in the studies discussed, Hydrogenation. All hydrogenations were performed on 50-mg. portions of t h e benzene eluates of samples 1 through 5. These portions were each dissolved in 30 ml. of n-heptane and hydrogenated for 12 hours at 200' C. using 0.5 gram of Raney nickel catalyst (7) and 3000 pounds per square inch of hydrogen pressure. The gaseous ieaction products were partially recovered by passing the gases from the hydrogenation bomb through a trap cooled by liquid nitrogen. The trap mas operated a t a pressure of 500 pounds per square inch to increase the recovery of condensable gases. The nonvolatile reaction products \T ere recovered by filtering the n-heptane solution through glass wool and blowing away the solvent a t 40' C. Urea Adductions. The urea adducts were prepared b y adding 5 nil. of methanol saturated with urea t o a solution of 150 mg. of a benzene eluate fraction in 30 ml. of a solvent composed of 4 volumes of benzene and 1 volume of methanol. After 24 hours t h e reaction mixture was filtered through sintered glass. T h e crystalline product was washed with 5 ml. of benzene, transferred to a 50-nil. beaker, and decomposed by heating with 10 ml. of distilled mater. The compounds which had formed the adducts with urea were extracted from the water with benzene and were recovered. The compounds n hich did not form adducts were recovered from the benzene layer of the filtrate after the urea and methanol had been removed by washing the filtrate several times with water. M a s s Spectrometry. The mass spectra were obtained with a Consolidated 21-103 mass spectronicter with modifications similar t o those described in the literature ( I , 6). The mass spectral data were corrected to a monoisotopic basis for carbon and hydrogen. For the isotopic corrections all ions were assumed to be hydrocarbons. .Isnonhydrocarhons were prwent, some of the corrections applied Iyere too large. The spurious peaks resulting from overcorrections were omitted from the spectra. After the isotopic correction, the data were normalized to the major peak, which was given the value of 100,000, This preserves the accuracy of measurement of the smaller peaks. I n the tables presented here, all peak heights were rounded off to one less figure and recorded in the grid form described by Clerc, Hood, and O'Neal ( 2 ) . I n this grid form the mass spectral peaks are arranged according to mass nuniber. Ions having the same mass number as hydrocarbon are found ions of the type (CnHSn+.)+ in row n and column z. For example, a n ion of a n even carbon number wax, or (C4r like (C2~H41-C-O-C~lH43) HMO,)+ has the same mass number as the hydrocwbon ion (CljHso)+ or (C4bH2x 4 5 - l o ) + and would be found in row n = 45 and column x = -10. Operating conditions for the mass spectrometer \yere: +
Ionizing current 45 pa. Magnet current 1.15 amperes (1.25 for spectrum of Table
1x1
Temperature Ionization chamber Inlet system Scan rate
250' C. 340' C. 3600 volts t o 250 volts in 35 minutes
Infrared Spectrometry. T h e infrared spectra were obtained with a Perkin-Elmer Model 21 spectrometer equipped with sodium chloride prism. The approximate concentrations of t h e samples in carbon disulfide arc' given in the legends of t h e figures. Sample and blank cells with silver chloride windows and a 3-mm. cell length !$-ere used. SAMPLE SOURCES A N D SIZES
The soils extracted for this study came from environments varying from swampy regions in Sumatra to arid regions in Texas. Sources of the soils and the sample numbers assigned the extracts are given in Table I. The benzene eluate fractions of the extracts were present in the soils in concentrations from 0.5 to 50 y per gram. The sizes of the samples available for study in this investigation ranged between 50 and 150 mg. Because of the limited sample sizes, none of the individual benzene eluates was studied by all the methods employed; however, the striking resemblance observed in the samples makes it probable that the data obtained for any sample or group of samples is iepresentative of all the benzene eluate fractions.
Table I.
Soil Samples
0
Location Dallas County, Tes. Tarrant County, Tex. Midland County, Tes. Sumatra Xiohrara County, K y o .
ti
Sumatra
Sample S o . 1
2
3 4
DISCUSSION A N D RESULTS
Samples 1 through 4 were used in the initial studies. The percentages of these samples removed by the four eluents from silica gel columns arc given in Table IJ. The infrared spectra of the benzene eluates of samples 1, 2, and 4 are shown in Figure 1. The vertical coordinates, representing per cent absorption, are offset so that all samples may be presented in one figure. The absorption maxima at 2.92 microns are probably caused b y OH groups. The strong maxima a t 5.76 microns result from ester carbonyl absorption. After saponification and acidification of these fractions, the carbonyl absorption shifts to 5.84 microns, corresponding to the acid carbonyl band. The absorption maxima in the region from 8 t o 9.5 microns are similar to those appearing in the
4 mg. per mi. of carbon disulfide
Concentration
Table 11.
Weight Per Cent of Samples Removed by Chromatographic Analyses
Eluent Sample Yo. 1 2 3 a
n-Heptane 1.8
Carbon tetrachloride 0.4
-
Benzene
\[ethanol
5 3
80 9 76 6
5.7 5 5 12 3 a 1 4 14 2 4 3 n-Heptrtnc fraction removed for previous stud? 1 8 8 4
spectra of esters of fatty acids. The maxima a t 13.9 microns are produced b y normal alkyl groups containing more than four carbon atoms. The chromatographic data for the hydrogenated benzene eluates of samples 1 through 4 are shown in Table 111. I n these analyses the n-heptane and carbon tetrachloride eluates are primarily saturated hydrocarbons. The chromatographic data and the infrared spectra of the n-heptane eluates (Figure 2) show that hydrogenation converts the major portion of the benzene eluates to saturated hydrocarbons. Data presented later in the paper indi-
74 2 72 7
I-niwovcred 11 6 10.6 0 0 8 8
cate that a niore complete coilversion of these eluates could have been obtained with a freshly prepared catalyst. The mas? spectra of the n-heptane eluates of the hydrogenated benzene
Table 111.
eluates are surprimgly similar considering the diverse origins of the samples. 4 typical example is the mass spectrum of sample 1 (Table IV). The larger peaks in this spectriiin are cornparablc in size to the peaks appearing at the same mass nurnberi in the spectra of this fraction of the other samples. The dternately high values occurring a t odd carbon numbers in the z = $2 column &howthat the normal paraffins of odd rarbon nuinher nre present in these fraction. in nmch higher concentrations than the even cnrlion number homologs. The l a r g ~T aluec in the z = - 6 column a t 27, 26, and 29 carbon atomq are parent pe:ikq of tetracyclic hjdrocxr-
Weight Per Cent of Sample in Chromatographic Analyses of Hydrogenated Benzene Eluates
Sample h-0.
Eluent Carbon t ctrachloride
Benzene
llethanol
4
Concentration
E
2 0 mg. per ml. of carbon disulfide VOL. 2 9 , NO. 8 , AUGUST 1 9 5 7
1155
I1 carbons, as does the spectrum in Table IV. Further proof that the benzene eluates contain sterols is provided by the facts that the original fractions give positive sterol color tests and that naturally occurring sterols contain 27, 28, and 29 carbon atoms, as do the tetracyclic compounds in the hydrogenated fractions. The volume per cent of the normal paraffins and parent sterol hydrocarbons-Le., steranes-in the n-heptane eluates of the hydrogenated benzene eluates are tabulated in Tables VI and YII, respectively. The concentrations presented were calculated from the
bons. The fragments in the z = - 7 column a t 26, 27, and 28 carbons are probably ions of the tetracyclic hydrocarbon molecules which have lost a methyl group, and the maxima a t 16 carbons in the z = - 6 and 7 columns are caused by ring systems. The similarities existing between the mass spectra in Table I V and of cholestane in Table V indicate that the tetracyclic hydrocarbons in the hydrogenated soil extract fractions are produced from sterols. The cholestane spectrum s h o w large peaks a t z = -6 a t 27 carbons, z = -? a t 26 carbons, z = -6 and -7 a t 16 carbons, and z = - 5 a t
-
Table IV.
Carbon Xumber, n
z:
-11
Mass Spectrum of n-Heptane Eluate of Hydrogenated Benzene Eluate Fraction of Sample 1
-10
-9
6 8
19 i5 12 8 8 7 7 8 5 7 c 6 6 5 6 8 6 45 4 2
2
1
8 6
5 5 4 3 4 4 4 5 5 5 7 5 5 5 5 6 71 3 2 2
52 40 34 39 33 33 50 45 25 20 19 19 29 17 16 95 16 205 19 4 5 2
Mass Series (t in C J L + . ) -6 -5 -4 -3 562 6 122 91 1791 .59 821 211 2392 .~ _ ~ . -~ 347 2518 82 941 483 1935 104 884 1472 880 471 109 116 761 361 756 397 419 227 1357 171 137 182 608 464 112 123 311 1077 159 187 232 210 104 463 330 273 1183 112 44 193 370 61 31 21 23 43 46 105 42 22 97 59 82 19 40 63 91 15 30 17 10 27 36 16 10 10 19 22 9 8 15 7 6 13 11 7 0 9 14 6 5 8 96 7 5 4 268 3 4 339 8 4 2 3 31 4 2 6 3 3 2 3 1 1 2 3 8
-7
'-8
4 5
9 10 11 12 i3 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
12 4 2 22 23 22 24 20 18 16 19 46 18 17 32 23 16 13 10 14 12 14 13 10 15 235 10
5 3
15 286 487 383 277 242 274 239 350 277 442 1892 208 140 488 111
~~
io
33 23 31 28 85 193 236 23 9 2
2 2
Table V.
0
6 7 8
n
-11
5 7 47 60 42
10
11
12
13
11
14 15 16
16 10
I7 18
19 20 21 22 23 24
25 26 27
1 156
11
7 4 5
3
2
- 10 6
38 24 22 34 22 14 7 6
12 8 11
4
2
5 2 2
.. .. .. ..
..
..
9
..
..
..
..
ANALYTICAL CHEMISTRY
-9 5 9 12 41 107 ..
94 84 66 41 42 89 58 18 40 60 12 7 6 2 2
10 31 7
-8
5 6 9 33 34 28 44 32 26 19 40 146 19 9 34 3 9 3
5 'i2 13 36
-2 439 706 745 725 264 244 120 67 42 38 32 23 29 23 15 13 1i 12 10 10
~
~~
-1 5729 3779 2111 1564 916 771
0 1589 1073 619 416 346 283 242 207 174 155 132 124 89 90 62 56 45 38 32 28 23 22 16 13 10 15 6 7 4 4
181
105 70 52 41 29 21 17 15 12 10 8 7 6 6 5 4 3 4 3 2 2 2 1
~~
~
~~
Carbon Sumber , n 4
sensitivity data of pure normal paraffins and cholestane. The paraffin concentrations which exceed 2% are probably accurate to within 5% of the astual concentration, but the sterane concentrations are based on a single reference compound and should be less accurate. In addition to the normal paraffins and steranes, a moderate concentration of pentacyclic hydrocarbons and a smaller concentration of hexacyclic hydrocarbons with parent peaks at 30 carbon atoms (see Table IV) in the z = -8 and - 10 columns, respectively, are found in all the hydrogenated benzene
6
6
G
5 15 4 5 2 2 2
+l
10000 5965 4081 1190 837 657 542 455 384 337 295 258 213 188 145 125 106 91 76 64 55 48 35 29 21 21
11
11 6 3
+2 7 8
9 5
11
28 17 10 6 4 3 3
4 21 9 126 15 51 11
37
8
31 6 30 8 25 5 18 3 7
~
Mass Spectrum of Cholestane
-3
-7
10 418 818 551 339 354 582 541 862 376 1344 10000 146 41 153 11 59 20 87 10 50 2103 40
7 91 146 186 176 176 896 568 215 311 138 4374 1370 41 9 6 4 31 11 9 20 16 2699
36 149 1515 1686 1466 i57i 1310 4429 763 221 134 111 1000 95 69 51 66 54 10 16 1
17 6
26
107 318 507 1439 1169 427 1103 113 71 16 24 17 5 7 760 41 4 3 2 1 11 4
3555 3565 1556 628 679 95 46 61 52 22 22 12
38 4 2
-2 231 797 406 203 155 61 29 106 10 7 7 93 19 4 6 16 1
-1 4329 1835 1329 830 331 103 26 27 28 26 9 9 5 4 3 1
1
0
2078 706 122 24
21
5i 21 9 9 7
15 8 5
4
37
8
10
a
4 3
2 1
2 1 ..
. .
1
+1
230 96 24 12 15
..
..
1 8 6 10 r 1
42 14
I
4 8 5
s
5 2
eluates. The relative sizes of the peaks at 29 and 30 carbon atoms in the z = -9 and -8 and in the z = -11 and -10 columns and the absence of large peaks caused b y polycyclic ring systems other than the sterol ring systems (at z = -6 and - 7 , n = 16) suggest that the pentacyclic, hexacyclic, and steranes have similar structures. Because Dauhen and Richards (4) have shown triterpenes to be intermediates in the biogenesis of steroids, it is possible that these polycyclic h j drocarbons are the hydrogenolysis products of triterpenes. If these compounds are derived from triterpenes, the z = -10 and n = 30 peaks in the mass spectra of these n-heptane eluates of the hydrogenated soil extract fractions are, to the authors’ knowledge, the first evidence of the existence of hexacyclic triterpenes that contain only carbon atoms in the ring sybtenis. The following estimated concentration- of the pentacyclic and hexacyclic conipounds were calculated by assuming the same parent peak sensitivity for these hydrocarbons and cholestane. Yoluine 70_in Sample S o . _ ____ Hldrocaikion
Pent:wyclic Hexacyclic
1 5 5 17
2 3 4 5 2 7 14 5 2 4 9 14 0 7 2 5 19
On the basis of the data obtained in the study of samples 1 to 4, the benzene eluates n-ere assumed to be composed of 11 axes. The infrared spectra shoned the samples to contain high concentrations of esters, and the hydrogenation products were similar to the hydrocarbons that could be obtained by hydrogenolysis of a wax such as beeswax. Adkins and coworkers (3, 8 ) have found t h a t alcohols and esters undergo hydrogenolysis to yield hydrocarbons. Their investigations show that primary alcohols yield hydrocarbons containing one less carbon atoiii than the alcohol, secondary and tertiary alcohols give hydrocarbons containing the same number of carbons a< the alcohol, and ethyl esters produce hydrocarbons containing one less carbon than the acyl group. An extension of these findings t o v a x esters gives the following general equations:
Table VI. Volume Per Cent of Normal Paraffins in n-Heptane Eluates of Hydrogenated Benzene Eluates by Mass Spectrometric Analyses
Carbon Atoms per 1Iolecule 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Sample 1
0 0 1 0
2 3 3 6
8 8
1 1 4 1 0 9 3 4 0.8 3.2 0.7 3 5 1 0 3 9 0 9
3 1 0.7 1.9
Total normal paraffins
2 0 3 0 3 1 4 0 6 3 7 11 4 2 1 6 5 9 1.4 3.8 1.1 4 3 1 3 4 7 1 0 3 8 0.8 1.5
4 0 . 4 42.8
3 4 0 2 0 3 0 4 0 3 1 0 1 8 0 8 0 5 3 3 1 7 0 9 0 6 4 7 3 6 1 3 1 2 5 9 5 8 1.4 1.2 5 . 6 . 5.5 1.2 1.1 7 4 5 2 1 5 1 4 6 4 4 9 1 2 11 7 5 5 5 0.8 1.0 3.1 51.5
45.8
Table VII. Volume Per Cent of Parent Sterol Hydrocarbons in n-Heptane Eluates of Hydrogenated Benzene Eluates b y Mass Spectrometric Analyses
Carbon A4toiiis in Molecule
Sample 1 2 3 B 3 8 0
27 28 29
Totd parent sterol hydiocnrlion.
2 3 4 4 2 1 3 1 9 7 0 2 3 2 2 102 4 1 5 4
166 214
7 T
0
:I
l~C--O--lI”
+ 6H2 catalyst -RH
and alcohols forming the esters. Kaxes of the type shonn in Equation 2 yield a paraffin, R H , and if the R”OH is a sterol, a tetracyclic hydrocarbon having the same number of carbon atoms as the sterol. The mass spectruni of the benzene eluate of sample 5 is shown in Table TIII. This mass spectrum is presented because the mass spectra of samples 1 t o 4 \T ere obtained only to a mass of approximately 500. For the loner mass range the spectra of ail the benzene eluates are similar. The mass range for sample 5 n a s extended becau-e the
-
R’H
2CH,
+ 2H20
+ 5H2 catalyst RH + R”H + CHa + 2H2O
n-here R and R’ are alkyl groups and R ” O H are secondary (or tertiary) alcohols. Waxes of the type shown in Equation 1 produce two paraffin molecules, R H and R’H upon hydrogenolysis, and these two paraffins contain, respectively, one less carbon atom than the acids
.+
io..
C,,H,,: C..+. .Q+
9 5
0
Ii--h--O--CHJL’
bon number range. These waxes are the chief source of the normal paraffins that are found in the hydrogenation products of the benzene eluate. The larger peaks in the z = -10 column at odd carbon numbers starting at n = 39 indicate that the even carbon number waxes are present in this fraction in higher concentrations than their odd carbon number homologs. The even carbon number waxes are found a t odd carbon numbers in Table VI11 because all ions in the spectrum are placed as hydrocarbons, and even carbon nuniber esters of aliphatic acids and alcohols have the same masses as odd carbon number C,H2,hydrocarbons. The wax molecules, because of the two oxygen atoms they contain, have three less carbons than their position in the spectrum xould indicate. The waxes in sample 5 range in size from 36 to 52 carbons. The large peaks in the z = -9 column between 16 and 34 carbon atoms are primarily rearrangement peaks of the wax acids. The mass spectra of known aliphatic esters h a r e similar peaks. For example, isoamyl stearate has a large peak at n = 21 and z = -9. A possible structure of the stearate rearrangement ion is
(1)
(2)
data obtained on samples 1 to 4 indicated that waxes with molecular weights in excess of 500 were the probable source of the saturated hydrocarbons found in the hydrogenation products of the benzene eluates. In Table VI11 the parent peaks of aliphatic waxes appear in the z = -10 column in the 39- to 55-car-
Because the niaxima in the z = -9 column in Table VI11 are a t odd carbon numbers in the n = 17- to 33-range, a major portion of the wax acids have even carbon numbers and range in size from 14 to 30 carbon atoms. The size of the peaks a t z = -9 and n = 19, 21, and 23 shows that the 16, 18, and 20 carbon number wax acids are the most abundant acids. Therefore, since both the even carbon number waxes and wax acids are predominant, the even carbon number wax alcohols must he more abundant than their odd carbon homologs, and the approximate size of the alcohols is indicated by the difference in carbon number between the nnues and the wax acids. Some of the ions nhich produce the large peaks in the z = -10 column and the smaller peaks in the z = -8, +2, and 0 columns a t 27, 28, and 29 carbon atoms probably yield, upon hydrogenation, the conipounds producing the maxima a t 27, 28, and 29 carbon atoms in the z = -6 column in the spectra of the hydrogenated fractions (Tables IV and XII). Some of the ions at 29 carbon atoms in the z = +2 and 0 columns may give, upon hydrogenation, the compounds that produce peaks a t 30 carbon atoms VOL. 29, NO. 8, AUGUST 1957
1157
t E
cn
z a LT
WAVE LENGTH, MICRONS Infrared spectra of urea-adducted fractions of benzene eluates of samples 5 and 6 and of beeswax
Figure 3.
Concentration
10 mg. per ml. of carbon disulfide
Table VIII.
Carbon Nnmber, n 4 5 6
I
8 9 10 11
12
13
14 15 16 17 18
19 20 21 22 23 24 25 26 27 28 29 30 31
32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
Mass Series ( z in CnHzn+ #)
z:
-11
-10
-9
2061 519 505 803 1311 718 629 548 577 385 533 303 458 728 288 329 447 176 199 197 223 183 238 66 60 28 52 19 21 12
111 47 107 376 223 236 255 144 101 112 93 177 128 462 146 1314 119 681 129 436 101 377 400 695 54 57 23 40 30
83 127 206 519 664 980 680 400 320 298 40 1 404 398 1326 446 4594 185 1956 141 965 132 480 127 137
49
9
90 55 c-c
9
12 5 4 4 4
104
42 205 33 148 36
..
212 42 174
48 49
, .
30
1 158
..
-7
-6
66 61 33 21 1 232 207 220 209
128 737 1424 1416 1135 944 837 511 361 562 455 187 166
32 228 266 363 468
111
97 174 147 102 52 41 55
..
70 16 71 51 51
81
is
11
19
73 31 8 8
2 2
1 .. ..
107
99 83 80 46 51 45 25 39
22 10 16
n
s5
14
5
11
12
4
329
218 150 130 143 162 821
81
81 87 119
118 88
86 89 -.
121 50 42 37 35 27 19
19 10 16 10 9
m
i
4 2
8
2
9
7
;3
9
9
11 6 6
4 3
2 2 ..
1
-4
-5
390 1458 1315 1155 1048 755 . .~ 48 1 334 260 303 255 191 131 160 151 183 67 54 34 32 35 29 19 16 10 10 6 0 5
4 4 3 4
324 515 510 443 335 235 124 96 184 150 139 153 125 119
57 57
42 -~
4i 55 53 38 30 64 70 23 12 11 9 9
37 i0 30 25 10 13 11
93 111
71 70
1
G 4
4
1
J
.. . . . . . .
G
1
a
1
.. .. .. ..
..
..
.. .. ..
..
, .
.. ..
r
0
+1
c2
1067 661 484 499 365 309 329 261 413 469 439 494 234 20 1 200 145 138
10000 8389 6653 3730 1739 902 672 537 439 400 330 263 272 186 230 140 125 94 83 71
3286 2632 5914 1823 705 513 433 402 334 315 251 520 228 1340 243 434 212 497 163 236 128 187 103 178 325 68 36 33 21 24 15 22 16
8385 4991 1029 738 657 673 611 553 45 1 363 332 365 455
73 59 144 174 3i5 262 199 154 106 91 88 99 94 161 115 174 119 138
109
123 137 117 68 69 35 45 25 24 16 19 12
, .
..
..
..
9 6
7..3
74 53 101 27 17
24 11 10 8
7 5 6 4 4 2 3 1
18 11 11 7
7
455 _. ~
714 219 388 183 229 137 16Y
240 115 171 78 31 35 17
19 10 11 9 7 8 5 3 5 3
118
105 92 106 138 199 399 64 30 38 20 20 14
18
12 18 9 11 c I
9
...
..
.. .. , .
.. . . . .
. . . .
...
..
...
, . .
. . ..
, .
,..
..
, . .
..
..
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i
ANALYTICAL CHEMISTRY
7
-1
6
c'
I I
18
9
-2
6
..
37
n
i
..
9
-3
1735 1084 603 366 440 292 253 226 187 168 116 114 83 71 59 49
132 20 r-
, .
-8
122 242 30
49
18
4i
50 51 52 53 54 55
Mass Spectrum of Benzene Eluate of Sample 5
..
...
100 W 0
z a I-
k
5
50
z a t r I-
s? C WAVE LENGTH, MICRONS Figure 4.
Infrared spectra of nonadducted fractions of benzene eluates of samples 5 and 6 Concentration
Table IX.
5 mg. per ml. of carbon disulfide
Possible Changes Produced In Some Components of Benzene Eluates by Hydrogenation
Ion Found in I
