method in limited numbers can be obtained on request from Director, Analytical Chemistry Division, Oak Ridge National Laboratory, P. 0. Box X, Oak Ridge, Tenn. 37830. Mechanical drawings for the construction of this apparatus were prepared on the basis of the prototype. Subsequently, two units were fabricated at ORNL from these drawings. Also, a 4-2792 polarograph and this 4-2942 apparatus were built from the ORNL drawings in Australia and perform very well (15). The satisfactory operation of these units shows
A substantial portion of the work to establish the conditions that result in optimum performance of this apparatus was done by W. L. Maddox, Oak Ridge National Laboratory. R. W. Stelzner and T. R. Mueller, ORNL, have also contributed to the project.
w. L. Belew, Oak Ridge National Laboratory, Oak Ridge, Tenn. (temporary assignment, Australian Atomic Energy Commission Research Establishment, Lucas Heights, N.S.W., Australia), personal communication, July 15, 1968.
R~~~~~~~for review February 26, 1968, Accepted March 5 , 1969. Research sponsored by the U S . Atomic Energy Commission under contract with Union Carbide Corp.
(15)
that the drawings are adequate for the duplication of this apparatus. ACKNOWLEDGMENT
Preparative Thin-Layer Chromatography and High Resolution Mass Spectrometry of Crude Oil Carboxylic Acids Wolfgang K. Seifert Chevron Oil Field Research Co., P.O. Box 1627, Richmond, Calif. 94802 Richard M. Teeter Chevron Research Co., Richmond, Calv. 94802 A carboxylic acid fraction of high interfacial activity isolated from a California crude oil was subjected to preparative thin-layer chromatography (TLC). The complexity of this fraction was reduced by TLC to about 1500 compounds, many of which belong to homologous series. Proof for absence of contamination was obtained. High resolution mass spectrometry studies of these carboxylic acids and derived trihydroperfluoroheptyl esters were performed. Based on exact masses of parent ions and fragments and on relative ion abundances, the presence of terpenoid, polynuclear saturated and mono- and polynuclear aromatic as well as naphtheno-aromatic ring structures is indicated. Comparison of acids and esters shows that the most abundant species contain 2, 3,4, and 5 saturated rings and fused polynuclear structures. The presence of many compound classes of carboxylic acids not discovered previously in petroleum is postulated.
A PREVIOUS paper ( I ) describes the isolation and separation of carboxylic acids from Midway Sunset 31E, California crude oil by countercurrent extraction, ion exchange, and silica gel chromatography. These techniques succeeded in separating phenols from carboxylic acids. However, the purest fractions were still too complex for direct identification by high resolution mass spectrometry. The thin-layer chromatographic (TLC) separation of petroleum products (2) in general, and, more specifically, of carboxylic acids (3) on silica gel has been described. This communication deals with preparative separation of the best fraction by thin-layer chromatography followed by high resolution mass spectrometry of carboxylic acids and derived fluoroalcohol esters (4). (1) W. K. Seifert and W. G. Howells, ANAL.CHEM., 41,554 (1969). (2) F. C . A. Killer and R. Amos, J. Inst. Petr., 52, 315 (1966). (3) J. C. Kirchner, “Techniques of Organic Chemistry, Vol. XII, Thin-Layer Chromatography,” E. S. Perry and A. Weissberger, Eds., Interscience, New York, pp 17, 243. (4) R. M. Teeter, ANAL.CHEM., 39, 1742 (1967). 786
ANALYTICAL CHEMISTRY
The fraction analyzed amounts to 5% of all carboxylic acids present in this crude oil and is representative of a larger percentage because of overlap of compound classes in various fractions. Because of the relative position (nonpolar portion) of the fraction in the total separation scheme, most of the structures postulated in this carboxylic acid fraction are free of heteroatoms. Besides the observation of a great number of compound classes of carboxylic acids not discovered previously in petroleum, the present study represents the first semiquantitative attempt to analyze a fraction of the carboxylic acids relative to a total virgin crude oil. EXPERIMENTAL Preparative Thin-Layer Chromatography. Reagents. The silica gel was Grade SG-DF-5 produced by the Camag Co. It contains 5 % calcium sulfate as a binder and an inorganic fluorescent indicator (3). The development solvent was a mixture of 0.5% distilled acetic acid (Baker and Adamson), 1% methanol (Mallinckrodt Chemical Co.), and 98.5 benzene (Baker and Adamson). The latter two were purified before use by passage through 28-200 mesh silica gel (Grace/ Davison). The ether (J. T. Baker Co.) was purified similarly with alumina (Merck Reagent Grade). All solvents were free of residue on evaporation. Separation. A slurry of 40 grams of silica gel in 84 ml of water was applied to eight glass plates (20.3 cm square), with a thin-layer spreader (Research Specialties Co.) producing a gel layer of 250-micron thickness. The plates were allowed to stand for 10 minutes at room temperature and thereafter for 1 hour at 105 “C and then in a desiccator for 2 hours. Carboxylic acid Fraction D-4 (53.9 mg), whose isolation from Midway Sunset 31E, California crude oil has been described in a previous paper ( I ) and whose separation is shown again in Figure 1, was dissolved in 0.5 ml of benzene and charged to the plates by the spot method. Development of the plates with the above-described solvent mixture was carried out by the ascending method and led to the formation of three separate zones. Zone 3 (Table I and
Zone 1
Table I. Preparative Thin-Layer Chromatography1 Ether Eluent Methanol Eluent SampleZ Sample Recovery, RCOOH Blank, RCOOH % Content3 mg mg Content'
mg 6.0
11 77
-
2 3
41.5 9.5 57.0
xx xxx
*.. -
X
0 0 9.9
-
4.3 6.0 5.6 15.96
Blank, mg
... ...
0 X
-
...
0
Total 8g6 9.9 1 Recovery from plates. Charged sample: 53.9 mg. 3 Infrared spectrometry; x = trace, x x = considerable amount, x x x = abundant. Zones were combined prior to elution. % Recovery = (wt-blank)/charge = (57.0-9.9)/53.9 = 87.4z. Agreement of sample with blank indicates than an insignificant amount of sample was recovered with methanol eluent.
Carboxylic Acids
Table 11.
-
m
16.4*,8
High-Resolution Mass Measurements Carboxylic Acid, Parent Peak Region
I o n Exchange W,eakly Basic Resin
-
IonExchange Slrongly Basic Resin
1
Ion Exchange -Weakly Basic Resln
A
Fraradlon* Sllica Gel
Tk
L ]This extract I l l represenls 3.54% 01 the tolal crude oil: 2.5% based on crude oil is RCOOH, see Tables 1, 11, a n d I I I o l r e f e r e n c e 1 . Percentages are based on total crude oil. Represents 5% of all RCOOH present in this crude oil,
Figure 1. Separation scheme Figure 1) was equally well separated in all eight plates and showed the strongest fluorescence under UV light. Recovery. The dry gel of each zone was scraped quantitatively from the plates and three batches of gel corresponding to the three zones of the eight plates were separately treated as follows: The gel was charged to a buret, the bottom of which had been plugged with cotton and ignited sand, both prewashed with methanol and ether. The gel of each zone was then eluted with 20 ml of ether while being stirred with a glass rod to maintain flow. Subsequent stirring of the gel in a flask with several portions of methanol totaling 80 ml per zone, filtration through a Grade F sintered glass funnel, centrifugation, and solvent evaporation led to the yields listed in Table I. Traces of acetic acid and water were removed by azeotroping with heptane and benzene. Eight blank plates were prepared, developed, recombined, and eluted in an identical fashion. The yields of contaminants are also listed in Table I. Esterification (4). Approximately 5 mg of the Zone 2 product was treated with 10 p1 of BF3.Etn0and 10 pl of 1,1,7-trihydroperfluoroheptanolat 50-60 "C for 15 minutes. Excess BFa and EtpO were removed in a gentle stream of NPat room temperature. The ester and unreacted alcohol were not miscible in the absence of BFs and EtnO and were separated with a microsyringe. Mass Spectrometry of Acids. A small sample of the acids isolated from Zone 2 of the thin-layer chromatography was placed in the cavity of the direct insertion probe recently
Formula
Calculated
Mass Measurement
Mass Error, ppm -~
264.1146 264.2086 266.130) 266.2244 268.1wo 268.1462 268.2394 270.1267 270.1610 270.2542 272.1765 274.1010 274.1926 276.1165 276.2081 278.2239 32.2870 384.m 4 384.3035 386 2244 386 2815 386 3177 388.2401 390. 1MO 414.2570 414.3128 414.3497 416.2722 416.36~ 418.2860 418.3789 420.3031 422.31% 424.3365 428.2697 430.2828 1
C18Hl,0~ 264.1150 CllHz,O~ 264.2089 CigH18Oz 266.1307 Cl,H,,Oz 266.2246 C17Hl,03 268.10)9 Cl,Hz,Oz 268.1463 C,,H,O, 268.2402 Cl,H180, 270.1256 Cl,HzzOz 270.1620 Cl,H,Oz 270.2559 C,,Hz,Oz 272.1776 C,,HI,Oz 274.0994 Cl,Hz,Oz 274.1933 Cl,Hl,Oz 276.1150 C,,HZ,OZ 276.2089 Cl,H,OZ 278.2246 Cz,H,,02 382.2872 CziHzsOz 384.2089 CziHioOz 384.3028 CZ~H,,OZ 386.2246 CzsH,gO, 386 2821 Cz,H,zOz 386.3185 CziH,zOz 388.2402 CZIHZ~OZS 3W.1654 CzqH,,Oz 414.2559 CziH,zO, 414.3134 Cz,H,Oz 414.3498 C 2 P H ~ , 0 ~ 416.2715 C,*HARO, 416.3654 C;;H;O; 418.2872 C,,H,O, 418.3811 CzqH,oOz 420.3028 CZ,H,~N~O, 422.3144 CZ~H,IOZS 424.3375 CioH,,OZ 428.2715 CziH38Nz0, 430.2831
-2 -1 +1 -1 -3 -0
-3 +4 -4 -6
-4
+6 -3 +5 -3 -3
Relative Peak Height'
Hydrocarbon --
-18 .I**
* ..t *
.*: *.* **** tt.
*
****
-1 +I +2 -1 -2
-2 -0 -4 +3
-1 -0 +2
-1 -3 -5 +l +1 -2
-4 -1
-4 -16 -2 -16 -14
10
3 9 2 9
8
0
1
-14 -12 +2 -10 -22 -8 -20 -6 -4
8
-12 -24 -10 -22 -10 -8 -20 -22 -22 -14 -8 -20
7 0 6 12
5 11
4 3 7
13 6 12 6 5 11 12 12
a
5 11
-6
A
-18
10 3 9 2 2 12
-4 -16 -2 -2 -22 -8
5
*** * Represents the most intense peaks within a mass range of
14. Relative heights were measured only for those peaks so marked. Z is derived from for the hydrocarbon which would be obtained on reduction of the carboxyl group to methyl. 3 R is the sum of rings plus double bonds and equals (2 - 2)p.
described by Gallegos (5) and examined in an AEI MS-9 high resolution mass spectrometer. Exact masses were measured by the peak matching technique as the sample was vaporized by slowly raising the probe temperature. The data are presented in Table 11. Mass Spectrometry of Esters. A small sample of fluoroalcohol ester was placed in the direct insertion probe, The mass spectrometer ion source was cooled to about 135 OC to slow external heating of the probe and consequent premature vaporization of the sample. A Leeds and Northrup Speedo(5) E. J. Gallegos, I. P. 30, Seventh World Petroleum Congress, Mexico City, D. F., Mexico, April 2-8, 1967. VOL. 41, NO. 6, MAY 1969
787
1
I
60
50
I
K, 50%
60%-
I
I
I
I
30
20 40%
IO
30%
I
I IO
I20
IO0
I
I
I
I80
I70
I60
I 0
70
I
60
90
80
I
I
I
I50
I40
I30
I20
TIME, MINUTES See T a b l e I l l . Areas were used t o compute f r a c t i o n o f sample vaporized.
t Boxed scans were examined i n d e t a i l .
*
Figure 2. Record of mass spectrometer monitor current during low resolution scans of fluoroalcohol ester
&la3
788
0.5 3.2 5.8
52 58 61
0.1 0.6 1.3
12.4
67
3.6
ANALYTICAL CHEMISTRY
E 21
90.2 96.5
100 102
109.6
108
48.7
max G recorder was connected to record the mass spectrometer monitor current which is a fixed fraction of the total number of ions and therefore can give a measure of the amount of sample being vaporized. With the probe in place, its temperature was slowly raised so as to maintain the total ion current at a usable level. Low resolution spectra were scanned periodically over a period of about 3 hours. The monitor record is shown in Figure 2. The curve was integrated by planimeter so that the fraction of the sample vaporized could be calculated for each mass spectrum, and this fraction is given along the top of the record in Figure 2. Table I11 lists the mass spectra, their times, probe temperatures, and the cumulative fraction of the total sample for each as well as the fraction of the sample that each of several selected scans represents. Two selected spectra are presented in Figures 3 and 4. With a new sample of ester in the probe, approximately the same temperature program was followed and high resolution mass measurements were performed by the peak matching technique on selected peaks with perfluorotributylamine as the mass reference. The high resolution data collected from the parent peak region are given in Table IV and from the fragment peak region in Table V. Fluoroalcohol Ester of 2-Norbornanecarboxylic Acid. The acid (-20 mg, Aldrich Chemical Co.) was treated with 1,1,7-trihydroperfluoroheptanol(-0.1 ml) and boron trifluoride etherate (-0.5 ml) for 4 hours at room temperature. The reaction mixture was diluted with two volumes of methylene chloride, washed with aqueous sodium bicarbonate and water and dried with anhydrous magnesium sulfate. The solvent was evaporated at about 80 "C in a stream of nitrogen. The residue, after several days at room temperature, deposited white crystals, one of which was removed and its mass spectrum was recorded at low resolution. The part of the spectrum above mje 360 is given in Figure 5.
Table IV. High Resolution Mass Measurements Fluoroalcohol Ester, Parent Peak Region Observed Mass
FoAki:,al
534.1437 536.1692 566.2024 570.1100 570.2353 576.1874 578.M36 580.2187 598.1731 598.2673 MNI. 1866 602.2028 W4.1238 W4.2190 606.2345 W8.1574 608.2501 610.1755 610.2683 612.1882 612.2&16 614.2011 634.2655 676.2216 676.3142 744 2836 744.3757 746.2992 746.3921 748.2249 748 2777 748.3148 748 4084 7%. 2391 750.3283 752.2517 752.3439 754.2659 7% 3595 756.2837 756 3759
Calculated Mass
Mass Measurement Error. oorn
M 4 w59 510.1428 534.1428 536.1584 566.2054 570.1428 570.2367 576.1897 578.2054 580.2210 598.1741 598.2680 WO. 1897 W2.2054 604.1271 wd. 2210 606.2367 608.1584 608.2523 610.1741 610.2680 612.1897 612 2836 614.2054 634,2680 676.2210 676.3149 744 2837 744 3716 746. 2993 746.3932 748.2244 748.2786 748.3150 748.4089 750.2401 750 3306 752. 2524 752.3463 754.2680 754.3619 756.2837 7%. 3776
-4 -4 t2 +3 -5 -5 -2 -4 -3
Relative
Hydrocarbon
Peak Hehht' -7-T -L
-4 -2 -1 -5 -4
It.
...t
-5 -3 -4 -2
it**
*.**
.**. ..
-4 12
. t
tl
t**
-3 t2
-7 -4 +1 -1 -0 -3 -0 -2 +l -1 -0 -1
.. ..
.s.
.. .. ..... 01.
.D
-3 -1 -3 -3 -3 0 -2
5 2 4 3 2 7 0 4 3
2 7 0 6 5 11 4 3 9 2 8
1 7
+2 0 -10 6 -4 3 -18 10 -4 3 -20 11 -6 4 -18 10 -4 3 -20 11 -18 10 -16 9 -2 2 -18 10 -14 8 -26 14 -12 7 -24 13 -10 6 -22 12 -8 5
..*e
-1
I\
-8 -2 -6 -4 -2 -12 12 -6 -4 -2 -12 +2 -10 -8 -20 -6 -4 -16 -2 -14 0 -12
.a.
.I.
...
Fluoroalcohol ester formula minus C?H2Fl2. See Footnote 1, Table 11. See Footnote 2,Table 11. See Footnote 3, Table 11.
RESULTS AND DISCUSSION
Isolation of Sample. The scheme of isolation and separation has been reported previously ( I ) , and part of it is repeated in Figure 1. Carboxylic acid fractions from Fraction D and Fraction B (Figure l), previously isolated by silica gel column chromatography and representing over 90% of all carboxylic acids in this California crude oil, were developed on silica-gelcoated plates on a microscale. Observation under ultraviolet light revealed that the cuts derived from the ion exchange fraction of lower polarity (B) were less uniform than those derived from Fraction D. The reason for this appears to be abundance of phenolic-type compounds in Fraction B as compared to Fraction D. One of the best separations was achieved with Fraction D-4 (Figure 1). The TLC screening of column chromatography fractions derived from Fractions D and B revealed decreasing separation efficiencies with increasing polarity; for example, the separation of Zone 1 from Zone 2 (Table I) was better in Fraction D-4 than in Fractions D-5 or D-6 (Figure l), and no separation was observed in Fraction D-7 or those of higher polarity [for further details of the analyses of these fractions, see Table I11 of the previous paper ( I ) ] . Fraction D-4 was selected for more detailed analysis because of optimum separation efficiency by thin-layer chromatography, and high interfacial activity, relative to the other fractions of D, as well as a minimum content of phenols, nitrogen, and sulfur compounds as indicated by the following analytical data. The number average molecular weight of this carboxylic acid fraction is 330, as determined by three independent methods ( I ) . An oxygen content of 10.8Oj, indicates that 90% of the oxygen is accounted for by carboxylic acids
Table V. High Resolution Mass Measurements Fluoroalcohol Ester, Fragment Peak Region Ester
Acid
Observed Mars Formuld' -401.0421 C,H,02
413.0422 413.0791 415.0194 415.0575 425.0413 425.0778 427.0577 428.0642 429.0367 429.0723 439.0577 439.0953 441.0717 442.0794 443.0859 453.0720 455.0874 463.0219 461.0871 48. 1012 480. 0979 480. I331 482 Ill5 505.1039 507.1193 5CQ. 1332 533. U66 533. 19M 535. 1519 537. 1680 549.1668 551 1822 575.1858
C5H102 C,H,,O C,H,03 C.H.0.
Ester Calculated Mass
MISS Measurement
401.0411 413.0411 413.0775 415.0203 415.0567 42225.Mil 425.0775 427.0567 428.0645 429,0360 429.0724 439.0561 439.0931 441.0724 462.0802 443.0880 453.0724 455.0880 463.m 467.0880 469.1037 480.0959
+2 +3
480.UB
+2
482 1115 505.1037 507.1194
+o
5CQ.UM 533. l3M
-3 +3
533. 1917
+1
535.1% 531. 1663 549.1663 551 1819 515. LBZD
'2 +3 +I
-3 -4
+7
-3 -1
Error, ppm
+4 -2
Frzction d Multipletz
Hydrocab
--+I z?
3
-1 -1 -1
+2
+1 -3
+I +1 -2 -1
-3 -1 0
+2 -0
+z
+5 -1
-5 -1
-1 +3
-2 -5 +4
0
-0
+o
0 1
1 1 0
2 2 1 1
-1 +1
1 0
-1
4
-3 0
2 1 1
+1
0
-3 -1 -9 -3 -1 -4 -2 -2
2 1 5 2 1 3 2 2
-1
-2
R4
-7
4
-5 -3 -7 -2
3 2 4
-5
3 2
2
3 2 4
See Footnote 1, Table IV. From measurement of peak heights at high resolution. See Footnote 2, Table 11. See Footnote 3, Table 11.
VOL. 41, NO. 6, MAY 1969
789
$388
510y
524
XI
a2 er
I
.
I
b
I
I ,
a
x 10
*-.JL-llruI.,
xl00
,,
I
I
I
200
300
400
I 500
I Mx)
mle *Probe tern perat ure 57 “C ,
Figure 3. Low-resolution mass spectrum fluoroalcohol ester Scan 2
191 h .a*v1
c W 4-
-c a3
.->
-m .a-
x loo
e
I
l
I
I
I
I
200
300
400
500
600
700
mle ‘Probe temperature 111°C
Figure 4. Low-resolution mass spectrum fluoroalcohol ester Scan 24 themselves, assuming one carboxyl group per molecule. Quantitative infrared analysis in carbon tetrachloride by a technique described previously (6) shows a phenol content of 3 mole per cent using the absorptivity of 2-isopropyl phenol as standard. A nitrogen content of 0.21% indicates that 5% of the acid molecules contain one nitrogen atom. Infrared frequency (6) at 3483 cm-1 as analysis using the free -NHthe analytical peak shows that less than 2 mole per cent of these nitrogen compounds are indoles substituted on the pyrrole ring andjor carbazole derivatives. A sulfur content of 0.82% points toward 8.5 mole per cent of the acids containing one sulfur atom. The separation of Fraction D-4 (Figure 1) by thin-layer chromatography was carried out on eight plates using a total of about 50 mg of material. A clear-cut separation of three zones with strong fluorescence in Zone 2 was observed. A major problem in preparative TLC separations for instrumental analysis of high sensitivity such as high resolution mass spectrometry is recovery of the sample free of contamination. Table I summarizes the recovery data (see also Experimental). It shows that the ether eluate of Zone 2 contains 77% of the sample with an abundance of carboxylic acids (IR). The yields obtained from blank plates (Table I) prove that this fraction is essentially free of contamination. Mass Spectrometry of Free Acid. Although the mass spectrum of the material isolated from Zone 2 was less complicated than that of the carboxylic acids prior to TLC, the sample still is a complex mixture. The range of molecular weights is from about 200 to about 700 with a maximum in the range 300-400. There is a peak at every mass over this wide range, and high resolution scans show each to be composed of from four to eight exact masses. Excluding odd masses, (6) W. K. Seifert, ANAL.CHEM., 41, 562 (1969). 790
ANALYTICAL CHEMISTRY
L
2
20435
rnle
Figure 5. Partial mass spectrum 1,1,7-trihydroperfluorohept yl norbornane-2-carboxylate which must be due to nitrogen compounds and carbon-13, there are about 1500 compounds present not counting the possibility of isomers. It is likely, however, that the types of compounds would be far fewer and that a limited number of homologous series would each be represented by a large number of members. The general appearance of the spectrum seems to confirm this. Figure 6 represents part of one of the low resolution scans of the free acids with only the even mass peaks shown. A periodicity of 14 is clearly evident, and two of the series are marked for clarity. The predominance of carboxylic acid functional groups, as discussed previously ( I ) , was confirmed by the occurrence of major peaks at m/e 60 and 74 in the mass spectra (7). (7) H. Budzikiewicz, C. Djerassi, and D. H. Williams, “Mass Spectrometry of Organic Compounds,” Holden-Day, San Fran-
cisco, 1967, p 214 ff.
A peak at m/e 191 was observed to be considerably larger than its higher and lower homologs. High resolution mass measurement showed it to be approximately 85% CUHS+ (obsd 191.1796, calcd 191.1800). This same fragment has been reported to be very prominent in the spectra of several terpanes isolated from petroleum (8-12) and several terpanes of known structure ( I I , I3--I5). It has usually been given (14) either of two structures (I, 11) which, in this sample, could originate W
PH2 H2c-s3 I
330
II
from different molecules or from different parts of the same molecule. In either case, it is good presumptive evidence for the presence of polycyclic di- or triterpenoids carrying, in the present case, a carboxyl group in some other part of the molecule. Detailed examination at high resolution (16,000-1 8,000) of selected parts of the mass spectrum yielded the mass measurements in Table 11. The group of peaks from m/e 264 through 276 covers one member of each homologous series present in this low mass portion of the parent peak region. Because the mass spectrometric sensitivities are not known, a true quantitative picture cannot be drawn, but relative peak heights can be used to give a general idea of the relative importance of the species detected. In order to avoid the interpretation of numbers as percentage composition, relative peak height in Table I1 is given on a scale of four with **** indicating the most intense peaks and * indicating the weakest. Each empirical formula was assigned a 2-number calculated + ~ the hydrocarbon that would from the expression C, H 2 % for be obtained on reduction of the carboxyl to methyl. From the 2, the number of rings plus double bonds (called R) was calculated (16) from the equation R = (2 - 2)/2. In addition to the group from 264 through 276, scattered other peaks were examined up to m/e 430 and their measured masses and empirical formulas also have been included in Table 11. All of the species in Table I1 contain at least two oxygen atoms and this was the criterion used for the selection of the listed formulas in those cases where mass measurement left some ambiguity. More peaks were found than are included, but the ones omitted are assumed to be rearrangement fragments, not molecular ions, because they are mostly hydrocarbon or single oxygen species. This overlapping of the parent peak and fragment peak regions can be eliminated, or at least greatly reduced, by conversion of the acids to esters of 1,1,7-trihydroperfluoroheptanol ( 4 ) . The mass spectra of
340
5 I
360
400
410
420
430
440
W e n masses only. 'Traction 0-4-TLC-2, see Figure 1.
Figure 6. Partial low-resolution mass spectrum of acid fraction
these esters greatly resemble those of methyl esters except that the peaks which contain the intact carbalkoxy group occur 300 units higher on the mass scale. Mass Spectrometry of 1,1,7-Trihydroperfluoroheptyl Esters. The mass spectra of the esters derived from the above acid fraction are spectacularly different from those of the free acids. Because of the expectation that the ester spectra would yield more information, a more extensive study was made of them than was felt worthwhile with the acids. A log of the mass spectra is given in Table 111. Eight spectra were selected (Scans 2, 6, 10, 14, 20, 24, 28, and 32) for detailed examination. Two of these (Scans 2 and 24) are given in Figures 3 and 4. The spectrum of Scan 2 (Figure 3) illustrates the relatively easy identification as parent peaks that can be made with fluoroalcohol esters. At the low mass end of the scale (up to about m/e 300) the spectrum consists of a mixture of hydrocarbon, acyloxy, and related fragments from the sample, with a few large peaks from unreacted fluoroalcohol-e.g., at m/e 231, 245, 282, and 313. At m/e 374 and 388 are two intense peaks resulting from the y-hydrogen migration and &cleavage of aliphatic ester moieties without and with an a-methyl substituent. These are analogs of the peaks at m/e 74 and 88 that occur in the spectra of similarly substituted methyl esters. Their formation is illustrated by the reactions [first postulated by McLafferty ( I Q ] shown below for the formation of I11 and IV for the methyl and fluoroalcohol esters, respectively. R>C&
(8) A. L. Burlingame, P. Haug, T. Belsky, and M. Calvin, Proc. Nar. Acad. Sci., 54, 1406 (1965). (9) . , W. Henderson, V. Wollrab, and G. Eglinton, Chem. Comm.,
- ;' C'R
Ot.
II
;hCH3 1968,710. 110) I. R. Hills and E. V. Whitehead, Nature, 209,977 (1966). ( l l j I. R. Hills, E. V. Whitehead, D. E. Anders, J. J. Cummins, and W. E. Robinson, Chem. Comm., 1966,752. (12) N. Danieli, E. Gil-Av, and M. Louis, Nature, 217, 730 (1968). (13) J. S. Shannon, Ausrr. J. Chem., 16, 683 (1963). (14) H. Budzikiewicz, J. M. Wilson, and C . Djerassi, J. Amer. Chem. SOC.,85,3688 (1963). (15) N, S. Wulfson, V. I. Zaretskii, and V. L. Sadovskaya, Tefruhedron, 22, 603 (1966). (16) K. Biemann, W. J. McMurray, and P. V. Fennessey, Tetruhedron Letters, 1966, 3997.
370 380 390 rn le
3
R>C*.tl.,
Ot. I ;c'ICH[''OCH2~CFz
),H
-
+
H.O+. I
H~C~~*OCH,
I l l ( m l e 74) R'LC I I +
c;
Hyc. I
H ~ C Q ~ ' O C H J C F),H~ I V (mle 374)
The presence of these rearrangement products says nothing about the nature of the esters beyond the a-carbon atom. Above m/e 388, all of the largest peaks are at odd mass until ~
~~
~
(17) J. A. Gilpin and F. W. McLafferty, ANAL.CHEM., 29, 990 (1957). VOL. 41, NO. 6, MAY 1969
791
m le *Esters of carboxylic acid fraction D-4-TLC-2, see Figure 1. **Calculated from data of Table I I I as described in text, Figure 7. Synthetic spectrum of fluoroalcohol ester parent peaks a homologous series of even mass parent peaks starts at m/e 496 and continues with 510, 524, 538, etc., up to about m/e 608. This series clearly defines the parent peak region. All of the spectra can be divided into three regions this way which we call, for convenience, the hydrocarbon fragment region, the ester fragment region, and the ester parent region. The actual boundaries of the regions, on the mass scale, increase with increasing probe temperature but the divisions remain fairly clear. On the assumption that all of the even mass peaks in the parent peak regions are, in fact, parent peaks representing unfragmented molecule ions, a synthetic spectrum was constructed as follows. The parent peak heights were read from the above listed spectra and multiplied by weighting factors derived from percentages listed in Table 111. A sum was then calculated for each nominal mass over all of the spectra and the sums were combined to yield the synthetic spectrum of Figure 7. Scan 2 was not used because it represents such a small fraction of the sample, but all of the parent peaks from each of the other seven scans were incorporated into the calculation of the synthetic spectrum. Figure 7 is an approximation of the “real” spectrum of the whole sample because seven representative spectra were used. Because the sensitivities of the various species making up the mixture are not known, a true composition cannot be determined; but an approximate average molecular weight of 676 was calculated assuming equal molar sensitivities. This average molecular weight for the 1,1,7-trihydroperfluoroheptyl ester implies an average molecular weight of 362 for the original acid fraction, in reasonably good agreement with the value of 330 obtained by titration and freezing point depression in camphor ( I ) and a value of 395 for the methyl ester obtained by vapor phase osmometry ( I ) . The major portion of the 0.6y0 of the sample represented by Scan 2 (Figure 3) consists of the homologous series of which m/e 510 is a prominent member. Exact mass measurement showed it to have the empirical formula C19H2202F12.Correcting this for the contribution due to the fluoroalcohol for the original acid yields an empirical formula of C12H2002 (Table IV). In every case where an empirical formula is given for an ion in an ester spectrum, it should be understood that this correction has been made. Hypothetical reduction of the carboxyl group to methyl would yield a hydrocarbon 792
ANALYTICAL CHEMISTRY
of formula C12H22 for which 2 = -2. Therefore, the number of rings plus double bonds (R)for the members of this series is two. The peak at m/e 496 is the lowest member of this series in the sample and corresponds to the ester of an 11carbon acid, CloH17COOH, which could be 1-, 2-, or 9-decalincarboxylic acid or possibly a bridged bicyclic structure such as a substituted norbornane, V. Additional alkyl substituents could account for the higher members of the series and the a-methyl group could be responsible for the large
peak at mle 388. Another way of explaining the molecular weights of the higher homologs is shown in Structure VI, affording an explanation for the large peak at m/e 401, the analog of the m/e 101 peak in the spectra of &methyl methyl esters (18). Another added methylene group between the carboxyl and the methyl branch could be responsible for the large peak at m/e 387, the analog of the peak at m/e 87 in the spectra of y-methyl esters (18). This interpretation is not required, however, because the fluoroalcohol esters of both cyclohexanecarboxylic acid (4) and 2-norbornanecarboxylic acid (Figure 5 ) exhibit intense peaks at m/e 387. It is concluded that saturated bicyclic acids are present and that the carboxyl group may be attached directly to the ring system but is probably mainly on an alkyl side chain. Scan 6 of the low resolution spectra represented about 9.2Y0 of the sample as measured by the monitor current (Table 111). The same peaks were present as in scan 2 but all were more intense. By Scan 10 (10.7% of the sample) it was evident that the molecular weight of the portion of the sample being observed was rising. The same homologous series was still predominant but the member at MW 510 was the lowest in mass, and the top end had moved up about two carbon atoms. By Scan 14 (17.070 of the sample), although the same 2ring esters were the most abundant single series, they no longer dominated the spectrum. Their odd-mass fragments were still large (for example, m/e 509, due to C12H~902) but ~
(18) R. Ryhage and E. Stenhagen, Arkiv Kemi, 15,291 (1960).
other kinds of even-mass peaks had grown, relatively, in the parent peak region of the spectrum. The most important were at m/e 550, 564, 578, and 592. Of these, m/e 578 was found to be CZ4H3002F12 and was therefore derived from C1iH2802. This empirical formula requires that R (rings plus double bonds) equal 3. A fragment at m/e 463 is the same as the largest peak, reported previously (4,in the spectrum of the difluoroalcohol ester of terephthalic acid. The meta and ortho isomers should yield ions of the same mass. The material responsible may not be native to the petroleum. Great care was taken to avoid contamination of the sample but if it had come in contact with a plasticized film, occasionally used in the laboratory to seal vials and flasks, a trace of phthalate plasticizer might have been introduced prior to esterification with the fluoroalcohol. Ester interchange would then have led to the observed species. In Scan 20 (15.70/, of the sample) the parent peak region extended from about m/e 562 to 694. The ester fragment region still started with the rearrangement peak at m/e 374 and overlapped the parent region to about m/e 579. The division was not distinct since there were large odd-mass peaks throughout the parent region, due, mostly, to contributions from alkyl-loss fragments and carbon-13 species, and, to a very small extent, to nitrogen compounds. The two-ring fragments (m/e 495, 509, etc.) were the largest, with the member of the series at m/e 551 the largest peak in the spectrum above m/e 388, but three-ring ions (m/e 578 and 592) had replaced two-ring parents as the most intense in this region except for the isolated m/e 598 peak. High resolution showed this peak to be caused by 86y0 C18H3602(no rings) and 14% C19H2a0z (an alkylnaphthalene acid). The large size and persistence of the 598 peak make it the largest in the entire parent region of the sample (Figure 7). The quantitative importance of the two-ring series of fragments does not imply a similar importance, in this molecular weight range, of two-ring compounds because multi-ring compounds can form two-ring fragments. In Scan 24 (Figure 4, 18.9y0 of the sample), the hydrocarbon fragment region begins to overlap the ester fragment region. The largest parent peak, by far, is the abovementioned m/e 598 peak. Two-ring fragments (495, 551, etc.) are the largest in their region of the spectrum but threering fragments (m/e 521, 535, 549, 563) are now prominent. At the low end of the ester fragment region, zero-ring peaks (m/e 401, 415, 429, 443) are also major contributors, probably evidence for the presence of carboxyl groups on side chains of varying length attached to the naphthenic nuclei. The parent region of Scan 28 (17.4% of the sample) showed no particularly outstanding homologous series. It extended from about m/e 636 to 842. The two-ring fragments covering the range m/e 467 to 551 were the largest peaks in the ester fragment region of from about m/e 400 to about m/e 635. The last scan of the series, Scan 32 (11.1% of the sample) carried the molecular weight up to about 960. Slightly outstanding was a series of peaks (m/e 802, 816, 830, 844, 858, 872) of which only a lower homolog was examined at high resolution. At m/c 746, 31% of the peak was due to C3oH4002 and 69y0 to C29H5202.The former formula requires 10 rings plus double bonds and the latter, three. A new major series of fragments ran, in steps of 14, from m/e 603 to 677 and another, from m/e 657 to 717. In both Scan 28 and Scan 32, a large peak was visible at mle 191, already discussed in connection with the spectra of the free acid mixture and presumably caused by pentacyclic triterpane acids. In the ester spectra, it cannot be
caused by neutral hydrocarbons because it does not appear until the parent peak region reaches about m/e 700 or higher which is appropriate for fluoroalcohol esters of acids in the 400+ molecular weight range. Gammacerene (MW 412), a hydrocarbon of this type, has been identified in a petroleum (10). The MW of an acid derived from it would be 442, and its fluoroalcohol ester, 756. High-Resolution Mass Measurement Data. A few of the fragments and parent peaks that were examined at high resolution have already been mentioned. Tables IV and V present the complete list except for peaks identified as containing carbon-13. About 5% of the nz/e 415 peak and about 6% of m/e 429 were found to consist of species containing three oxygen atoms, C 4 H 5 0 3and CBHi03,respectively, after subtraction of the fluoroalcohol contribution. No C6H~03was found at m/e 443. The observed species are probably acyl ions and are most likely caused by diacid monoesters in the original acid fraction. Genesis of a peak at m/e 429 of the correct composition could come about from the diester as shown. t
-
t
0 0 P 111 II H(CF~),CH,OG-C-OCH~(CF,),H C-C-OCH~(CF~),H 0
mie 429
C4and C 5 diacids have been reported in Green River Shale (19, 20) but would probably be too polar to appear in this
fraction as free acids because this fraction is the least polar portion of Fractions D and D-4. Consequently, if the C 4 H 5 0 3 and C 5 H 7 0 3fragments do arise from alkyl-oxygen cleavage as shown above, the species in the original oil was probably a halfester. A difluoroalcohol ester could form by esterification and trans-esterification. The nature of the alcohol portion of such an ester is not known. Alternatively, 4- and 5-keto acids could be the molecular precursors of these two fragment ions, but the evidence is insufficient to allow a positive statement. In either case, it can be concluded that there are present small amounts of acids containing carbonyl functions in the 4- and 5-positions but at concentrations too low to be visible as a doublet by IR, as would be expected for half-esters, Table V contains several peaks which mass measurement demonstrated to have only one oxygen atom. Even so, their presence is consistent with carboxylic esters, as the lightest of these, 14% of the small peak at m/e 413, is a higher homolog of a rearrangement peak previously found ( 4 ) in the mass spectrum of the fluoroalcohol ester of decanoic acid. Also in Table IV is a peak at m/e 533 whose exact mass fits that of Cz2HZ8O2Fll, formed by loss of a single fluorine atom from C22H2802F12. This loss of a fluorine atom from a molecular ion has been previously reported (4) and occurs quite commonly. It is responsible for the fragment of m/e 435 in the spectrum of the ester of 2-norbornane carboxylic acid shown in Figure 5 . Structures I and I1 were given earlier as possible structures for the terpenoid fragment at m/e 191. If either of these isomeric structures carried a carboxyl group in place of one methyl, the corresponding ester fragment would appear at m/e 535; and the unesterified fragment formula would be C14H2102.This species was detected (Figure 4) and is listed in Table V.
(19) A. L. Burlingame and B. R. Simoneit, Nature, 218,252 (1968). (20) P. Haug, H. K. Schnoes, and A. L. Burlingame, Science, 158, 772 (1967). VOL. 41, NO. 6, MAY 1969
793
Table VI. Comparison of Fluoroalcohol Ester Spectra Relative Peak Heights Octanoic Decanoic Dodecanoic acid' Scan 24 mle acid' acid' (m/e 514) (m/e 598) Parent (m/e 458) (m/e 486) peak 100 100 100 1002 387 374
730 945
293 303
317 308
1293 1903
From Reference (4). After subtraction of 1 4 z due to ester of CI,H~,OZ. 3 For m/e 598 species to be stearate ester, fragment peaks should be larger to be consistent with lower homologs.
Table VILA. Observed Parent Peak Molecular Formulas from High-Resolution Mass Spectra of Carboxylic Acid and Its Fluoroalcohol Ester Z = f 2 t 0 -6
"1
'2
Carbxylic Acid
R'/ 0
Formulas
Exact masses measured in the parent peak region are given in Table IV. The same relative intensity scale of four was used for the esters as was previously used for the acids (Table 11). Two coherent regions were examined in the ester spectrum, from m/e 600 through 612 and from m/e 744 through 756. As described above, the major part of the peak a t m/e 598 is due to an 18-carbon saturated acid. The most common acid that fits this description is stearic acid but the evidence of the fragment-peak relative heights indicates that this is probably not the principal structure present. The m/e 598 peak reached its maximum height in Scan 24. The heights of the 598 peak and two lower fragments (m/e 387 and 374) were measured arid are compared, in Table VI, to similar peaks in the spectra of three previously reported ( 4 ) straight chain esters. On the basis of the heights of the m/e 387 and 374 peaks of octanoate, decanoate, and dodecanoate fluoroalcohol esters, one would expect the same peaks from stearic acid to be considerably larger than those that appeared in Scan 24. Consequently, it is concluded that the C18H3602 acid must be largely branched although the presence of some stearic acid is not ruled out. It is interesting to note that the branched chain C18 acid observed here has not been reported previously (21-24). Table VI1 presents a summary of acid and ester empirical formulas and tentative structural types. The information derived from Tables 11, IV, and V was grouped together in Table VI1 by 2 and R. The listing of possible structural types warrants some comments; in particular, it must be emphasized that only structural types, not detailed structures, are presented. The main evidence is drawn from the empirical formulas derived from exact mass measurements on the molecular ions. This approach, however, usually requires that a choice be made in each case between possible alternatives. The structures that are given in Table VI1 were selected for a variety of reasons which are summarized below. The presence of significant fragments in the mass spectrum supports the tentative structural types assigned to formulas with R = 0, 1, 2, 4, and 5. In particular, the six-membered ring was chosen for R = 1 because m / e 441 (acid fragment formula C7H1102)is the most intense peak in its homologous series. The prominent peak at m/e 191 supports the terpenoid structure for R = 5.
(21) J. Cason and D. W. Graham, Tetrahedron, 21,471 (1965). (22) G. Eglinton, A. G. Douglas, J. R. Maxwell, J. N. Ramsay, and S. Stallberg-Stenhagen, Science, 153, 1133 (1966). (23) R. F. Leo and P. L. Parker, ibid., 152,649 (1966). (24) A. L. Burlingameand B. R. Simoneit, ibid., 160, 531 (1968). 794
0
ANALYTICAL CHEMISTRY
Fluoroalcphoi Ester Relalive
Formulas'
C17H340Z
Peak
U
1 C,,HjzOt
C16H3?02 18H3102
**
C,,H,Oz
POSSlble Strudural Types,
Aiiphallc Carboxylic Acid, Branched or Straight Chain.
Cl,H,OZ
1 2
1
pz't$&, -**'
References6 21-28
RDCaoH R ~ C O O H
08cooH
R
d
pJCOOH
See Footnote 2, Table 11. See Footnote 3, Table 11. See Footnote 1, Table 11. See Footnote 1; Table IV. 6 The positions of the carboxyl and alkyl substituents are arbitrary; carboxyls are not necessarily directly attached to rings but may be on an alkyl side chain. Two skeletal structures found in petroleum. 'Two independent sets of ester peak heights are represented covering the ranges m/e 600-612 (esters of C19H2602-C19H3802) and m/e 744-756 (esters of C ~ Q H ~ ~ O ~ - C ~ O H ~ O O ~ ) . In addition, a few types of acids have been identified previously from petroleum or petroleumlike sources. Analogy with these acids was used in Table VI1 for the cases of R = 0, 1, 4, 5, and 7. Pertinent references are listed in the last column of Table VII. Furthermore, hydrocarbon types known to occur in petroleum were the analogical bases for assignment of structure for R = 0, 1, 2, 4, 5 , 7, 8,9, 10, 11, 12, 13,and 14. Aswith the acids, references are listed in Table VII. The most speculative set of structures are those with a hydrophenanthrene structure (R = 3 and 6). The greater abundance of phenanthrene over anthracene in this crude oil (to be proved in future papers) and in a representative petroleum (32) governed this choice. With the exception of R = 13 and 14, all of the assignments made in Table VI1 are supported by additional methods of molecular spectrometry (UV, IR, fluorescence, NMR, and MS based on fragment ions and combined with gas chromatography) of hydrocarbons prepared by reduction of these acids. This work will be reported in the future. (25) K. A. Kvenvolden, J . Am. Oil Chem. SOC.,44, 628 (1967). (26) J. Cason and A. I. A. Khodair, J. Org. Chem., 32,3430 (1967). (27) M. Blumer and W. J. Cooper, Science, 158, 1463 (1967). (28) I. MacLean, G. Eglinton, K. Douraghi-Zadeh, R. G. Ackman, and S. N. Hooper, Nature, 218, 1019 (1968). (29) J. Cason and K.-L. Liauw, J. Org. Chem., 30, 1763 (1965). (30) H. M. Smith, U. S. Dept. of Interior, Bureau of Mines, Bulletin 642 (1968). (31) L. P. Lindeman and R. L. LeTourneau, Proc. Sixth World Petrol. Cong., Frankfurt, Germany, Sect. 5, 1963, p 281. (32) B. J. Mair and J. L. MartinCz-Picb, Proc. Am. Petrol. Inst. Sect. III, 42, 173 (1962). (33) P. Haug, H. K. Schnoes, and A. L. Burlingame, Geochim. Cosmochim., Acta., 32, 358 (1968).
Table VII-B. Observed Parent Peak Molecular Formulas from High-Resolution Mass Spectra of Carboxylic Acid and Its Fluoroalcohol Ester 2 = -8 to -16
Table VII-C. Observed Parent Peak Molecular Formulas from High-Resolution Mass Spectra of Carboxylic Acid and Its Fluoroalcohol Ester 2 = -18 to -26 2'
d
-18
10
Possible Slrudural
-C10H4001
*'
R&COOH
32
RC-OOH
M,33
Lipids are generally considered to be a major source material for petroleum (30, 36, 37). If such is the case, the carboxylic acids of petroleum, more closely related chemically than hydrocarbons to many of the lipid precursors, should more closely reflect the composition of the source. This postulate cannot be adequately checked with the current data because the quantitative aspects of the study are not well enough developed. However, it is interesting to note that two-, three-, four-, and five-ring saturates are major contributors to this acid fraction. If the analogy of acid structure with known hydrocarbon structure is valid, then a major portion of the pentacyclic saturated acids should be terpenoid. The structures postulated in this paper for carboxylic acids in a California petroleum will be given further support in future reports on separation and molecular spectrometry of hydrocarbons derived from these acids. CONCLUSIONS
Preparative thin-layer chromatography on silica gel has been shown to be an effective method of simplifying the complexity of crude oil carboxylic acids of high interfacial
(34) (35) (36) (37)
References'
-I
'IP",I",
R-COOH
mypar'
H. M. Smith, J. Am. Oil Chem. Soc., 44, 680 (1967). W. Carruthers and D. A. M. Watkins, Chem. Znd., 1963, 1433. E. D. McCarthy and M. Calvin, Nature, 216,642 (1967). Sir R. Robinson, Nature, 212, 1291 (1966).
-24
13
-26
14
Ca,Hz,02
activity and has resulted in the isolation of 5% of the carboxylic acids present in Midway, Sunset 31E, California crude oil. High resolution mass spectrometry studies of this fraction and derived fluoroalcohol esters allows the conclusion that the fraction contains about 1500 compounds, many of which belong to homologous series. The most prominent carboxylic acids are saturated 2-, 3-, 4, and 5-ring compounds, possibly derived from steroids or polycyclic terpanes. In addition, carboxylic acids of similar carbon skeleton are present but contain aromatic and naphtheno-aromatic rings, most of which have not been reported before. Small amounts of half-esters of succinic and glutaric acids with unknown alcohols are present. Eighteen-carbon carboxylic acids are present and we conclude, from the relative sizes of certain fragment peaks, that both branched and straight chain structures are present. A large proportion of the carboxylic acid groups are on side chains appended to naphthene or aromatic rings and not on the rings directly. ACKNOWLEDGMENT
The authors express their appreciation to P. E. Monson and A. D. Alderman for technical assistance. RECEIVED for review October 23, 1968. Accepted February 3, 1969. Presented in at the Gordon Research Conference on Geochemistry, New Hampshire, August 1968.
VOL. 41, NO. 6,MAY 1969
795
