C19, and CZOisoprenoids, identical with those of authentic farnesane, pristane and phytane, respectively, add to the certainty of the identification. The completely straight line gas chromatograms of the procedure blanks showed that the samples were not contaminated during analysis. Because some seemingly ambiguous identifications of isoprenoid hydrocarbons have appeared in the literature, it must be emphasized that reliable identification of these compounds can be accomplished only if the gas chromatographicmass spectrometric analysis is preceded by an extensive wet chemical fractionation. It was found in this laboratory, that (a) saponification to remove acids and esters, followed by (b) molecular distillation to remove nonvolatiles, (c) dilute HC1 extraction to remove bases, (d) elution chromatography on alumina to remove nonhydrocarbon compounds, and (e) on silica gel to remove aromatic compounds, and finally (f) fractionation of the resulting hydrocarbons into straight and branched/cyclic fractions with molecular sieves accomplished this end. The molecular distillation step appears to be beneficial because it has been found (14) that high molecular weight components in chromatographic samples reduce the separation efficiency and may retard the elution of the com-
ponents from alumina or silica gel beyond their normal point of emergence. These same considerations may also apply to capillary gas chromatography, which, even without undue sample compositional problems, is sufficiently difficult to optimize for tandem mass spectrometry. The mass spectra (Figures 3-5) demonstrate that by careful performance of all these steps, quite rigorous mass spectral identifications can be obtained for isoprenoid hydrocarbons even where isomers are known to give similar spectra. ACKNOWLEDGMENT
The authors wish to thank Donald Baker of Rice University, Houston, Texas, for supplying the rock sample; Eugene D. McCarthy of the University of California at Berkeley for providing the farnesane and phytane standards which he synthesized and Harold C. Urey of the University of California at San Diego for his advice. RECEIVED for review January 8, 1968. Accepted February 26, 1968. This study was supported by the National Aeronautics and Space Administration, under research grants NsG-541 and NGR-05-009-043.
Analysis of Tall Oil by Gel Permeation Chromatography Teh-Liang Chang Research Seruice Department, Central Research Division, American Cyanamid Co., Stamford, Conn. 06904
THECONVENTIONAL ASTM method ( I , 2) for determining resin acids in tall oil is by titration following preferential esterification of fatty acids. The fatty acid is determined by substracting the resin acid content from total acid content. This method requires a skillful technique and is often subject to a large error. \ A more sophisticated method studied extensively recently employs gas chromatography (3, 4). The acids are esterified and then separated into the individual acids. The high separability by gas chromatography unnecessarily complicates an analysis where only total resin acids and fatty acid data are required. An accurate and simple method for tall oil analysis certainly is desirable. It is known that separation by the gel permeation chromatography (GPC), technique developed by Moore (3, is based on molecular size (6). GPC, then, appears t o be a promising tool for tall oil analysis. Because the major constituents are fatty acid, resin acid, their dimers and trimers, it should be possible to separate them by their sizes. This possibility was also implied in a review article by Bartosiewicz (7). A careful investigation was therefore undertaken. A similar work was also performed by Zinkel(8). An agreeable result
(1) Am. SOC.Testing Mater., D 803 ASTM Std., Part 18 (1965). (2) Am. SOC.Testing Mater., D 1240 ASTM Std., Part 20 (1967). (3) F. H. Max Nestler and D. F. Zinkel, ANAL.CHEM.,39, 1118 (1967). (4) R. G. Ackman, J . Gas Chromatog., 1(6), 11 (1963). ( 5 ) J. C. Moore, J . Polymer Sci., 2,835 (1964). (6) R. L. Pecsok and D. Saunders, Sep. Sci., 1, 613 (1966). (7) R. Bartosiewicz, J. Paint Technol., 39, 28 (1967). (8) D. F. Zinkel, U.S.D.A., Madison, Wisconsin, private communication, Dec. 1967.
was obtained even though different column substrates and eluents were used. It was found that the GPC method has several advantages over other methods. The analysis is carried out at room temperature and a complete analysis of acid components is made in a single experiment. Tall oil is analyzed for fatty acids, resin acids, fatty acid dimer, resin acid dimers and resin acid trimers. No pretreatment of the sample, such as esterification, is required. The original constituents of the sample are not destroyed and can be recovered for subsequent identification by established techniques. EXPERIMENTAL
Apparatus. A multicolumn unit was used. It consisted of four 4-ft X 0.25-inch stainless steel columns, two of which were packed with Bio-bead S-X2 gel and the other two, with Bio-bead S-X8 gel (Bio-rad Laboratories). Another type of column substrate examined was Sephadex LH-20 gel (Pharmacia Fine Chemicals). A differential refractometer (Waters Associates) was employed as the detector. The temperature was maintained at 30.0' =t0.1 C by a Lauda Circulator Model K-2 (Lauda Instruments Division of Brinksman Instruments, Inc.). Tetrahydrofuran (THF) was the eluent and the solvent. The eluent flow rate was maintained at 40 ml/hr with a Lapp LS-20 Microflo Pulsefeeder Pump (Lapp Insulator Inc.). A pressure release valve was attached to the system to regulate the operational pressure. The flow rate was first controlled by the operational and then finely adjusted by a needle valve. The operational pressure was approximately 8 psi/ft of column. Reagents. Tall oils, fatty acids, resin acids, fatty acid dimers, wood rosin and gum rosin were supplied by the Arizona Chemical Co. All other chemicals were obtained from Eastman Kodak Co. O
VOL 40, NO. 6, MAY 1960
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n
w L
-In
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70 I60
40 50
30
-
20
-
7 6 5 -
I 11
4 -
90
I IO
100
I
I
0.2
0.3
0.4
I20
K
0.5
130 rnl ELUTION VOLUME
I
I
0.6
\
UNCORRECTED FOR THF CORRECTED FOR THF
2 I
I
I
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I
0.7
Figure 1. A gel permeation chromatogram of tall oil
Figure 2. Relationship of acid carbon chain length (log #C) and distribution coefficient K
RESULTS AND DISCUSSION
The column substrate selected for this study was a crosslinked nonionic polystyrene polymer, Bio-bead S gel. All of the injected acid sample was fully recovered and no delay in elution time was noticed. With the crosslinked polydextran polymer, Sephadex LH-20 gel, a strong absorption effect was found. The acid sample was either retained permanently in the column or eluted out much later than a hydrocarbon of comparable molecular size. The upper molecular weight exclusion limit of Bio-bead S-X8 gel is approximately 1,000, Because the molecular weights (mol wt) of fatty acids and resin acids are about 300, their polymers, except dimers, were poorly separated and the polymer peaks were concentrated on the high mol wt side of the chromatogram. This poor separability, however, was improved by adding another Bio-bead S-X2 column, which has an upper exclusion limit of approximately 3,000 mol wt. The upper and lower exclusion limits of the prepared columns were determined by eluting 0.1 polystyrene and 0.1 % H 2 0 solutions. The polystyrene used has a molecular weight of about 20,000. The HzO gave a negative refractive index peak when T H F was used as the eluent. When a tall oil sample was eluted through the GPC column, it was separated into several peaks. A chromatogram of tall oil is shown in Figure 1. In order to identify the
Table I. Distribution Coefficients and Effective Carbon Chain Lengths Compound K #Ccalc #Cobs Acetic acid 1.0 2.7 Valeric acid 0.88 5.7 Nonanoic acid 0.72 9.7 Myristic acid 0.58 14.7 Stearic acid 0.49 18.7 0.49 18.7 Oleic acid Linoleic acid 0.49 18.7 0.49 18.7 Fatty acid Fatty acid dimer 0.24 37.2 Abietic acid 0.66 11.4 Levopimaric acid 0.66 11.4 Dehydroabietic acid 0.66 11.4 Resin acid 0.66 11.4 Resin acid dimer 0.41 23.2 Resin acid trimer 0.28 33.8
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ANALYTICAL CHEMISTRY
resolved components more efficiently, each separated peak was denoted by its distribution coefficient, K , which is its relative elution volume between the elution volumes of the higher and lower exclusion limits (9). The distribution coefficient is a specific characteristic of a peak and can be reproduced within an average error of iO.01 unit. It is usually independent of flow rate, column length and solute concentration. It is, therefore, a better expression for a peak than the elution volume or the elution time. It was found that CISfatty acids, resin acids or fatty acid dimers gave a well-defined peak. The distribution coefficient ( K ) of these fatty acids was 0.45, of resin acids 0.66, and of fatty acid dimer 0.24. Because the separation by GPC is based on the molecular size, it was expected that resin acids or fatty acids with similar molecular structure should generally possess similar distribution coefficients. This assumption was found to be true in this study (see Table I). The distribution coefficients of abietic, levopimaric acid, and dehydroabietic acids were 0.66, while those of stearic, oleic, and linoleic acids were 0.49. It was reported by Hendrickson and Moore (10) and Chang (11) that a linear relationship exists between the logarithm of the molecular size and its elution volume, or distribution coefficient. The molecular size was expressed as effective carbon chain length, #C. When a correction of +3.0#C unit was also made for the hydrogen bonding between T H F and acid, the data for acids agreed with that for hydrocarbons of comparable molecular size. These results were employed to predict the identity of some of the unknown peaks observed in this study. A standard curve was prepared from acetic acid (#C = 2.7), valeric acid (#C = 5.7), nonanoic acid (#C = 9.7), myristic acid (#C = 14.7) and stearic acid (#C = 18.7). Their distribution coefficients, K , were plotted against the logarithm of #C. A linear relationship was obtained after the correction for hydrogen bonding with T H F was made (Figure 2). When the distribution coefficients of fatty acids and fatty acid dimers were projected into the extrapolation of the (9) M. J. R. Cantow, "Polymer Fractionation," Academic Press, New York, 1967, pp 138. (10) J. G. Hendrickson and J. C. Moore, J. Polymer Sci., 4, 167 (1966). (11) T. L. Chang, Anal. Chim. A m , 39, 519 (1967).
I
I
Table 11. Analysis of Synthesized Samples Sample
#
I
J RESIN ACID
11 F A T T Y ACID 111 FATTY ACID
0.5
1.0
1.5
DIMER
2.0
PER CENT
Figure 3. Concentration calibration curves for resin acid, fatty acid and fatty acid dimer
1 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16
Fatty acid dimer Resin acid added found added found
Fatty acid added found 50.0% 70.0 75.0 85.0 25.0 20.0 10.0 5.0 99.0 99.5 99.8 99.9 1.0 0.4 0.2 0.1
49.3% 69.7 74.5 85.4 25.6 20.4 10.2
4.8 99.0 99.4 99.8 100
0.9 0.4 0.3 0.1
25.0% 25.4% 25.0% 10.0 20.0 20.3 5.0 10.0 10.3 20.0 5.0 4.9 25.0 50.0 49.6 70.0 69.7 10.0 5.0 85.0 84.6 10.0 85.0 85.6 1.0
1.0
0.5 0.2 0.1 99.0 99.6 99.8 99.9
0.6 0.2 0 99.1 99.6 99.7 99.9
25.3z 10.0
5.2 19.7 24.8 9.9 5.2 9.6
Table 111. Analysis of Tall Oils, Wood Rosin and Gum Rosin standard curve, the #C observed for fatty acid was 18.7 and for fatty acid dimer was 37.2. The linearity of the standard curve was followed up to #C = 37. When the K for resin acids (0.66) was projected into the curve, the observed #C was 11.4. This value is much smaller than the general molecular formula of resin acids, ClaH&OOH, would indicate. However, considering the tricyclic structure of most resin acids, this appears to be a reasonable value. Sample #18 is a polymerized resin acid which contained no fatty acid. When this sample was eluted through the GPC column, it was separated into a major component and two minor components. The major component (K = 0.66, #C = 11.4) was a resin acid monomer. The two minor components were higher mol wt substances than resin acid and were eluted earlier than resin acid. Their distribution coefficients were K = 0.41 and K = 0.28. Their effective carbon chain lengths observed from the standard curve in Figure 2 were #C = 23.2 and #C = 33.8, respectively. It can be seen that they are approximately twice and three times the size of resin acid monomer. It is reasonable, therefore, to conclude that these two minor components are resin acid dimer and resin acid trimer. The relationship between the sample concentration and the recorder response was investigated for fatty acids, resin acids, and fatty acid dimer. The calibration curves were prepared by plotting the sample concentration vs. the peak height as shown in Figure 3. A linearity was followed up to 2% for each acid, or to 10 mg because the sample size was 0.500 ml. The sensitivity was 5 pg of acid or lods% of the solution; the average deviation, or the error of reproducibility, was 10.5%. Because pure resin acid dimer and resin acid trimer were not available t o us during this study, they were not studied. However, it was assumed that a linearity would also be followed between the peak height and the sample concentration for resin acid dimer and resin acid trimer. The calibration curve for resin acids was used for the determination of resin acid dimer and resin acid trimer. The detection limit for fatty acid in rosin was studied by adding various concentrations of fatty acid to resin acid solution and determining the amount of recovery. As little as 0.2% of fatty acid in resin acid was determined with an average deviation of 10.1%. The detection limit of resin
Sample Fatty # acid % 17 18 19 20
21 22 23 24 25 26 27 Wood rosin Gum
rosin
...
...
1.2 31.8
Fatty acid dimer
.,. ...
Resin Resin Resin acid acid Others acid dimer % trimer % 94.4 82.0 93.6
5.6 11.8 4.4
,.. 4.3
... ... ... ... ... ... ... ...
...
1.9 0.8
68.2 99.1 96.8
...
15.8 7.0 2.8
,..
32.3 21.9 34.2 74.6
0.6 12.6
5.8
7.0
0.3
...
96.1
1.2
...
2.4
.. .
...
91.0
2.9
...
6.1
0.9
... 100 51.9 71.1 62.4
.:.
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acid in fatty acid was also studied. As little as 0.2 =k 0.1 % of resin acid was detected (see Table 11,samples #9-16). A set of samples was prepared to examine the validity of this analytical method. These synthesized samples contained various concentration of fatty acid, resin acid and fatty acid dimer. The analytical results are listed in Table 11, samples #1-8. A satisfactory analysis was obtained and all the added components were recovered within an average deviation of 10.5%. Eleven tall oil samples were analyzed by the established GPC method. Because the major concern of this study was to establish an analytical method rather than to study the nature of the sample, only the major components were determined and tabulated in Table 111. The minor components can also be analyzed by amplifying the chromatogram. This will be useful when the main concern is kinetic study, composition determination, or structure identification. Some of these studies are in progress and will be reported in the near future. The main natural sources of resin acid are tall oil, wood rosin and gum rosin. Because it would be helpful to have VOL 40, NO. 6, MAY 1968
991
an analytical method which could be used for all of them, a typical wood rosin sample and a typical gum rosin sample were examined. It was found that wood rosin consisted of 96.1 of resin acid monomer, 1.2% resin acid dimer and about 3 % of other components. Gum rosin consists of 91.0% of resin acid, 2.9% resin acid dimer and a very high concentration, 6.1 % of other components. These preliminary studies indicate GPC to be a promising technique for resin acid analysis.
ACKNOWLEDGMENT The author acknowledges Arizona Chemical Company for supply of chemicals and consultation on composition of samples. Received for review November 7, 1967. Accepted January 18, 1968. This work was supported, in part, by Arizona Chemical Company. Presented at Division of Analytical Chemistry, 155th Meeting, ACS, San Francisco, Calif,, April 1968.
Gas Chromatographic Estimation of Individual Xylene Isomers in Straight-run Naphtha Cuts P. L. Gupta and Pradeep Kumar Indian Institute of Petroleum, Dehradun, India
SEPARATION of the xylene isomers by gas chromatography using both capillary and packed columns has been the subject of numerous publications (1-9). Among the packed-column separations, the best results have been obtained with a modified Bentone-34 column (9) [bentonite clay of formula A1203 (SiO&HzO, reacted with dimethyldioctadecylammonium ion]. The Bentone column, though quite suitable for separation of CS isomers in wholly aromatic mixtures, has limited selectivity for separating the aromatics from the corresponding saturates which have about the same boiling points, and therefore, it cannot be used for estimating the aromatics in petroleum fractions and other similar samples. Also, this column gives a poor separation of o-xylene from isopropyl benzene. The present study concerns the estimation of the individual xylene isomers in straight-run naphtha cuts boiling up to 150' C in the presence of all the usual saturates. The separation is achieved by studying the behavior of several combinations of a Bentone column with various other columns to determine the conditions which are suitable for satisfactory resolution of the following three pairs of compounds : n-Nonane and Ethyl Benzene. n-Nonane (bp 150.8' C) has been assumed to be the typical paraffin in the xylene range, and its separation from the lowest boiling Cs aromatic, ethyl benzene (bp 136.2' C), was taken to represent the separation of the saturates from the aromatics. Because of nonavailability of branched and cycloparaffins of the xylene range, their actual separations from ethyl benzene could not be studied, but care was taken to ensure that the n-nonane-ethyl (1) D. H. Desty, A. Goldup, and B. H. F. Whyman, J . Insf. Petrol. (London), 45, 287 (1959). (2) J. S . Dewar Michael and J. P. Schroeder, J . Am. Chem. SOC., 86, 5235 (1964). (3) Albert Zlatkis, Su Yu Ling, and H. R. Kaufman, ANAL.CHEM., 31, 945 (1959). (4) L. C. Case, i. Chromarog., 6 , 381 (1961). (5) J. Van Rvsselberae - and M. Van Der Stricht, Nafure, 193, 1281 (1962).16) - . J. Gas Chromaton., _ . . , M. Van Der Stricht and J. Van Rvsselberge. 1(8), 29 (1963). (7) J. V. Mortimer and P. L. Gent, Nature, 197,789 (1963). ( 8 ) Samuel F. Spencer, ANAL.CHEM., 35,592 (1963). (9) Edward W. Cieplinski, Ibid., 37, 1160 (1965). .
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benzene separation was sufficient to avoid interference with the ethyl benzene peak by any cycloparaffin, with a boiling point of about 150' C, whose retention time would be between that of n-nonane and ethyl benzene on the selective column combination used. p and rn-Xylene. This is the most difficult pair to separate. A good separation of the p- and rn-xylene pair normally implies a good separation of ethyl benzene from the next higher boiling isomer, p-xylene (bp 138.4' C), with the exception of the existence of very severe conditions when the boiling point elution order for the Cs aromatics is completely upset. The separation of o-xylene (bp 144.4' C) from the p - or rn-isomer does not pose any problem on any of the stationary phases because of the large difference in boiling points. @Xylene and Isopropyl Benzene. This pair is considered because of the tendency of the isopropyl benzene peak to interfere with that of the o-xylene on Bentone column and on most of the polar stationary phases. This difficulty may not arise for well fractionated cuts, because isopropyl benzene is well separated from o-xylene (bp 152.4' and 144.4' C, respectively). In actual practice, however, the presence of small amounts of Cs aromatics cannot be completely eliminated in xylene cuts from naphtha fractions. Other CS aromatics are unlikely to interfere with any of the xylenes. The separation of saturates from the aromatics with the same boiling point (first pair) can easily be achieved on a polar stationary phase, whereas for the 2nd pair, a modified Bentone column (9) is perhaps the most promising. Therefore, a suitable combination of the Bentone column with a polar one is likely to give the desired separation. But such a combination may not give any separation of the 3rd pair at all, because on many polar phases, the elution order of these two compounds is the reverse of that on Bentone. The behavior of the individual column combination, therefore, is studied and its selectivity properly adjusted to achieve a good separation of the 3rd pair without seriously impairing the other two separations. EXPERIMENTAL Apparatus. A Jobin-Yvon gas chromatograph equipped with a flame ionization detector system and a 2.5-mV Sefram
