+
to the conjugated ion (CH&N=CH-CH=S, due to loss of hydrogen from the ion of mje 104. Another peak at rnje 87 is due to loss of CH2-CH2, an important process in the elec\S/
tron impact fragmentation of thioesters (7), while the peak at
+
mje 72 probably represents the ion (CH3)2N-CHrCH2. Analytical Results. Clearly interpretable spectra were obtained from a minimum of 0.5 pg of acetylcholine bromide (2.2 nmoles) using thk heated inlet system, first generating the sample vapor in the closed reservoir and then scanning the spectrum within a few minutes of allowing the vapor to effuse into the ionization chamber. Thermal decomposition of the tertiary amine began to occur at temperatures above 180 “C, but below 160 “C, little dissociation of the quaternary salt took place. It was found convenient therefore to rapidly heat the sample oven to about 190 “C while maintaining the reservoir, orifice leak, and ionization chamber at 180 “C. Under these operating conditions, maximum peak heights were obtained for the mje 131 and 87 ions with little change in peak height with time. However, the peak heights of the rnje 71 and 58 ions dropped significantly after 2 to 3
minutes in the reservoir. Furthermore, it was found that when a 1 solution of acetylcholine chloride was progressively diluted down to 0.05 %, the diagnostic tertiary amine peak heights were directly proportional to the amount of material contained in the 2.5 pl of solution withdrawn only in the range 2 to 10 pg (11 to 55 nmoles) as shown in Figure 1. On the other hand, the heights of the peaks at m/e 50 and 52 due to CH3Cl+.were unaffected by either temperature, dilution or the time that the vapors remained in the reservoir and as also shown in Figure 1, were linearly dependent on the amount of acetylcholine chloride down to the detection limit of about 0.02 pg (that is approximately 0.1 nmole). Thus, the methyl halide provides a suitable substance for the quantitative estimation of total choline derivatives in a mixture. Of the halides, the iodides are most favorable because iodine is monoisotopic and therefore gives only one peak at mje 142 rather than two in the ratio 1 :0.324 and 1 :0.979 for CHICl and CHI Br, respectively, and because the total ionization cross-section of molecules is directly proportional to their molecular weight (8).
RECEIVED for review May 13,1968. Accepted July 8,1968. (8) J. H. Beynon, “Mass Spectrometry and Its Applications to
(7) W. H. McFadden, R. M. Seifert, and J. Wasserman, ANAL. CHEM., 37, 560 (1965).
Organic Chemistry,” Elsevier Publishing Co., Amsterdam, 1960, p 239.
Determination of Double Bond Positions in Polyunsaturated Fatty Acids by Combination Gas Chromatography-Mass Spectrometry Walter G . Niehaus, Jr.,’ and Ragnar Ryhage Department of Chemistry and Laboratory for Mass Spectrometry, Karolinska Institute, StockhoIm, Sweden Fatty acids and hydroxy fatty acids which contain one to five double bonds were oxidized to the corresponding polyhydroxy acids with permanganate or osmium tetroxide. The polyhydroxy acids were converted to polymethoxy methyl esters with dimethylsulfinyl carbanion and methyl iodide. These derivatives were analyzed by gas chromatography-mass spectrometry. Characteristic fragmentation between methoxylsubstituted carbon atoms allowed the determination of the positions of the methoxyl groups, and thus of the positions of the double bonds in the original fatty acids. Ambiguities in fragment identification were eliminated by the determination of the elemental composition, through the use of a peak-matching accessory of the LKB-9000 single focusing instrument, which allows determination of the molecular weight with an accuracy of 10 parts per million. With another modification of the instrument, which will simultaneously record the intensities of three different m/e values as a function of time, it was possible to use this method to determine the compositions of mixtures of derivatives of monounsaturated fatty acids, which were not resolved by gas chromatography, with an accuracy of &lo%. THEDETERMINATION of the positions of ethylenic bonds in multiply unsaturated fatty acids is a formidable problem. Classical approaches involve the isolation of each fatty acid in pure form, and oxidative cleavage at the unsaturated centers with ozone or permanganate, followed by identification of the 1840
ANALYTICAL CHEMISTRY
fragments by gas chromatography. The total recovery and quantitation of all cleavage products is difficult, as the short chain fragments have appreciable water solubility and high volatility. Although these methods are fairly satisfactory for monounsaturated fatty acids, they become less useful for compounds with a number of double bonds. Positional isomers of polyunsaturated fatty acids may be identified by a comparison of their gas chromatographic retention times with those of standards. Ackman (1) has employed a low polarity silicone polyester phase for the separation of a number of isomeric polyunsaturated fatty acid methyl esters derived from marine oils. The determination of double bond position by mass spectrometry has thus far been of little practical importance. Positional isomers of monounsaturated fatty acids give very similar spectra, which do not indicate the position of the double bond (2). Catalytic deuteration of double bonds leads to extensive isotope incorporation and to exchange of deu1 Present address, Department of Biochemistry, Pennsylvania State University, University Park, Pa.
(1) R. G. Ackman, J. Gas Chromatogr., 4,256 (1966). (2) B. Hallgren, R. Ryhage, and E. Stenhagen, Acta Chem. Scand., 13, 845 (1959).
followed by conversion to the methyl ethers for analysis by gas chromatography-mass spectrometry. The method should also be applicable to unsaturated keto- or epoxy-fatty acids, which could be converted to mono- or di-hydroxy unsaturated fatty acids, and possibly could be extended to the localization of the positions of ethylenic bonds in other multiply unsaturated acyclic molecules. A preliminary account of some of this work has been published (11). Figure 1. Mass spectrum of the derivative of palmitoleic acid
terium (3), so that the spectra of these derivatives do not indicate the position of the double bond. Analysis of the spectra of derivatives produced by reduction of the double bond with deuterio hydrazine ( 4 ) is complicated by partial H-D exchange reactions (5), and by rearrangements within the mass spectrometer (6). Epoxidation of an unsaturated fatty acid followed by treatment with sodium iodide yields a mixture of two isomeric keto fatty acids. The mass spectra of these derivatives contain peaks which allow one to determine the position of the double bond in the original acid (7). The mass spectra of dihydroxy fatty acids, produced by permanganate oxidation of unsaturated fatty acids, contain a major peak corresponding to fragmentation between the carbon atoms bearing the hydroxyl groups (8). None of these methods has been applied to fatty acids containing more than one double bond. The combination of gas chromatography with mass spectrometry offers many advantages for the analysis of double bond positions. The samples need not be isolated in pure form, for mixtures of unsaturated fatty acids may be assayed directly, the separation occurring on the gas chromatograph column. For such analysis, the unsaturated fatty acid must be converted to a derivative which may readily be gas chromatographed, and which undergoes characteristic cleavage giving rise to fragments which indicate the position of the double bonds in the original compound. McCloskey and McClelland ( 9 ) have determined the double bond position for a number of monounsaturated fatty acids, with the o-isopropylidene derivatives of the diols obtained by oxidation of the double bonds. The spectra of these derivatives contain several peaks which indicate the position of the isopropylidene group. The method also appears to allow one to distinguish between cis- and trans-double bonds. The application of the method to fatty acids containing more than one double bond has not been reported. A similar study has been performed with unsaturated hydrocarbons (10). We have developed a method which allows localization of double bond positions in fatty acids containing one to five double bonds, as well as in unsaturated hydroxy acids. The method involves oxidation of the double bonds to glycols, (3) N. Dinh-Nguyen and R. Ryhage, J. Res. Inst. Catalysis, Hokkaido University, 8, 73 (1960). (4) N. Dinh-Nguyen, R. Ryhage, and S. Stallberg-Stenhagen, Ark. Kemi, 15, 433 (1960). (5) N. Dinh-Nguyen, R. Ryhage, and S.Stallberg-Stenhagen,ibid., 18, 393 (1961). (6) R. Ryhage and E. Stenhagen, "Mass Spectrometry of Organic Ions," Academic Press, New York, 1963, p 399. (7) G. Kenner and E. Stenhagen, Acta Chem Scand., 18, 1551
(1964). (8) R. Ryhage and E. Stenhagen, Ark. Kemi, 15, 545 (1960). (9) J. McCloskey and M. McClelland, J. Arne). Chem. Soc., 87,
5090 (1965). (10) R. Wolff, G. Wolff, and J. McCloskey, Terrahedron, 22, 3093 (1966).
EXPERIMENTAL
Apparatus. Gas chromatography-mass spectrometry was performed with the LKB 9000 instrument. A column of 1 % SE-30 on Gas-Chrom P was operated at a temperature between 200" and 250 "C. The ion source temperature was 270 "C. The energy of the bombarding electrons was kept at 70 eV. The ion outdrawing potential was increased from a normal value of 4 V to about 8 V, which gave a higher intensity to the diffuse peaks caused by metastable ions. Magnetic scanning was used and the mass spectra were obtained in about 5 seconds. Reagents. The unsaturated fatty acids used in these experiments were acquired from the following sources : hexadec-9-enoic (palmitoleic) acid, octadec-6-enoic (petroselenic) acid, eicos-l l-enoic acid, octadeca-6,9-dienoic (linoleic) acid, eicosa-5,8,11J4-tetraenoic (arachidonic) acid, and 12-hydroxy octadec-9-enoic (ricinoleic) acid from Sigma ; octadec-9enoic (oleic) acid, octadeca-9,12,15-trienoic (alinolenic) acid, octadeca-6,9,12-trienoic(y-linolenic) acid, and eicosa-5,8,11,14-17-pentaenoicacid from The Hormel Institute; octadec-11-enoic (vaccenic) acid from Fluka. 9-Hydroxy octadec-12-enoic acid was a generous gift from G. J. Schroepfer, who isolated the acid from Strophanthus oil. Osmium tetroxide and methyl iodide were purchased from Merck. Dimethyl-sulfoxide was distilled from calcium hydride under reduced pressure. Dimethylsulfinyl carbanion solution (-2M) was prepared from dry dimethylsulfoxide and sodium hydride (12). The carbanion solution was stored frozen in test tubes under an atmosphere of nitrogen and was thawed immediately before use. Procedure. ALKALINE PERMANGANATE OXIDATION.Fatty acids containing one or two double bonds and unsaturated hydroxy acids were routinely hydroxylated with alkaline permanganate (13). The fatty acid (0.1 to 100 pmoles) was dissolved in 0.2 ml of 0.25N NaOH and diluted with 1 ml of ice water. 0.2 ml of 0.05M K M n 0 4 was added and after 5 minutes the solution was decolorized with SO,. The fatty acid derivatives were extracted with CHC13-CH30H (2 :1). The CHCI3 phase was removed, dried (MgSOJ, and the solvent was evaporated. OSMIUMTETROXIDE OXIDATION.Fatty acids containing three or more double bonds did not yield the desired products when treated with alkaline permanganate, and were therefore oxidized with osmium tetroxide (9). The fatty acid (0.1 to 100 p mole) was dissolved in 1 ml of dioxane-pyridine (8:l) and 0.1 ml of a solution of OsOl in dioxane was added. After one hour at room temperature, 2.5 ml of methanol and 8.5 ml of 1 6 z Na2S03in water were added, and the mixture was allowed to stand for an additional hour. After centrifuging to remove Na2S03, the supernatant solution was diluted with four volumes of methanol and filtered. The filtrate was evaporated to dryness and suspended in 2 ml of methanol. Chloroform (4ml) was added, the suspension was filtered, and the solvent was evaporated. PREPARATION OF POLYMETHOXY DERIVATIVES FROM THE POLYHYDROXY FATTY ACIDS. The methylation was carried
5z
(11) W. Niehaus and R. Ryhage, Tetrahedron Lett., 5021 (1967). (12) E. Corey and M. Chaykovsky, J. Amer. Chem. Soc., 84, 254 (1962). (13) K. Wiberg and K. Saegebarth, ibid., 79,2822 (1957). VOL 40, NO. 12, OCTOBER 1968
1841
Figure 2. Mass spectrum of the derivative of 9-hydroxy octadec-12-enoic acid out essentially as described by Hakomori (14). The polyhydroxy fatty acid was dissolved in 1 ml of dry dimethylsulfoxide; 1 ml of dimethylsulfinyl carbanion solution was added, and the tube was flushed with nitrogen and allowed to stand at room temperature for 15 to 20 minutes. Methyl iodide (0.2 ml) was added dropwise; the tube was flushed with nitrogen and allowed to stand an additional ten minutes. Ten milliliters of ether was added, followed by 5 ml of water. The tube was shaken twice and the ether layer was removed. The mixture was extracted two additional times with ether. The combined ether extracts were washed several times with water to remove most of the dimethylsulfoxide, dried (MgSOJ, and evaporated. The residue was dissolved in hexane and injected directly into the gas chromatograph-mass spectrometer. The polyhydroxy fatty acid derivatives were not isolated in pure form, but were converted directly to the polymethoxy methyl esters, Although yields were not determined, the size of the gas chromatographic peaks indicated a nearly quantitative conversion of the polyunsaturated fatty acids to the polymethoxy derivatives. The gas chromatogram showed no significant impurities with retention times approximating those of the derivatives, although some peaks were seen with short retention times. These compounds produced mass spectra characteristic hydrocarbons and presumably arose from polymerization of methyl iodide. These impurities, which are less polar than the derivatives, are eluted from a column of silicic acid by 5 % ether in hexane. The derivatives may then be eluted with increasing concentrations of ether. The derivatives, purified in this manner, may be introduced into the mass spectrometer via the direct probe, and give spectra virtually identical to those obtained from the gas chromatographic effluent. RESULTS AND DISCUSSION
I. Mass Spectra of Derivatives of Monounsaturated Fatty Acids. The mass spectrum of 9,lO-dimethoxy methyl hexadecanoate, derived from palmitoleic acid (Figure 1) is typical of the spectra obtained from derivatives of monounsaturated acids. No molecular ion peak is seen, but significant peaks are present at mje 299 (M - 31), m/e 267 [M - (31 32)], and m/e 235 [M - (31 2 X 32)], presumably representing the loss of (-OCHJ and successive losses of the elements of methanol. The principal fragmentation occurs between the methoxylsubstituted carbon atoms, Cg and Cia, giving rise to the ion fragments designated A (m/e 201) and B (m/e 129). These primary ion fragments subsequently lose the elements of methanol, fragment A (m/e 201) yielding the fragments at rnje 169 and 137, and fragment B yielding the fragment at mje 97. Metastable ions characteristic for these secondary fragmentations are present in the spectrum. A much less significant primary cleavage is seen alpha- to
+
(14) S. Hakomori, J. Biochem. (Tokyo),55,205 (1964).
1842
ANALYTICAL CHEMISTRY
+
Figure 3. Mass spectrum of the derivative of ricinoleic acid the methoxyl-substituted carbons (between Cs and Cg) resulting in an ion fragment at rnje 173, and a secondary peak at m/e 141 (173 - 32). Cleavage also occurs between Cloand Cll, resulting in a very small peak at m/e 245, and a larger peak at m/e 213 (245 - 32). The location of the double bond in a monounsaturated fatty acid may therefore be deduced from the peaks representing the primary fragments A and B in the general formula:
A-B I I
CH3-0-C-(CH2),-CH
CH-(CH&-CH,
I!
I
I
0
OCH3
OCHI
(1)
The dimethoxy derivatives of threo- and erythro-9,lO-dihydroxyoctadecanoic acids were prepared and the mass spectra were measured. No significant differences could be observed in the two spectra. Thus the method does not allow one to assign the cis- or trans-configuration of the double bond in the original fatty acid. 11. Mass Spectra of Unsaturated Hydroxy Fatty Acids. The spectra of 9,12,13-trimethoxy methyl octadecanoate, derived from 9-hydroxy octadec-12-enoic acid, and of 9,10,12-trimethoxy methyl octadecanoate, derived from ricinoleic acid, are shown in Figures 2 and 3. The peaks representing M (31 32) and M - (31 2 X 32) are present at m/e 325 and 293, but of lower relative intensity than in the spectra of the dimethoxy derivatives. Fragmentation between the methoxyl-substituted carbon atoms is again prominent, and cleavage alpha- to the pair of methoxyl-substituted carbons occurs in the 9,12,13-trimethoxy methyl octadecanoate, giving rise to a peak at m/e 159. Cleavage also occurs alpha to the other methoxyl-substituted carbon, yielding peaks at mje 129 and 201 in the respective spectra. The structures of the derivatives of these monounsaturated-monohydroxy fatty acids may thus be deduced from these characteristic fragments. 111. Mass Spectra of Derivatives of Fatty Acids with 2 to 5 Double Bonds. The spectra of the derivatives of linoleic acid, a-linolenic acid, y-linolenic acid, arachidonic acid, and eicosapentaenoic acid are shown in Figures 4-8. The peaks seen for the derivatives of the monounsaturated fatty acids at 32), and M - (31 2 X 32) are exM - 31, M - (31 tremely small or absent in the spectra of the derivatives of polyunsaturated fatty acids. The principal fragmentation occurs between the methoxyl-substituted carbon atoms, as was seen for the simpler derivatives. Secondary fragments arise from successive losses of the elements of methanol, as verified by the appearance of the corresponding metastable ions. Cleavage alpha- to the initial and final pairs of methoxyl-substituted carbons is also significant. The structures of the derivatives of straight chain methyleneinterrupted polyunsaturated fatty acids, having the general formula :
+
+
+
+
Figure 6. Mass spectrum of the derivative of 7-linolenic acid
Figure 5. Mass spectrum of the derivative of a-linolenic acid
Figure 7. Mass spectrum of the derivative of arachidonic acid
were elucidated in the following manner. Fragmentation between any pair of methoxyl-substituted carbons gives rise to an ion fragment (A,) with the general formula:
substituted carbons would have mje values of 44 (102 0 ) (14 X 4) 15 = 115, 44 (102 X 1) (14 x 4) 15 = 217,44 (102 X 3) (14 X 4) 15 = 421, and 44
0
I
+
+ +
+
+
mje = 59
I
+
+
+
x
+ +
+ 14a + 102y + 58 (3)
OCHa OCH3
or to an ion fragment (B,) with the general formula: mje
For any particular polyunsaturated fatty acid, the values of The value of y may be zero or greater, depending on the position of the cleavage. Each primary fragment in the spectrum-i.e. the highest member of a series off, (f 32), [f (2 X 32)], etc.-can be fitted by trial and error into one or the other general formulas, and the values of a and b may be determined by solving the appropriate equation, with only positive integers for a, b, and y . Calculating the expected values for the fragments A1, A S ,etc. and for the fragments Bl, Bzretc., and comparing these values with the mass spectrum allows one to determine the total number of methoxyl groups, and thus to elucidate the complete structure of the derivative. Such data have been summarized in Table I for the derivatives examined. To illustrate the procedure, the data from the spectrum of the derivative of arachiconic acid may be treated as follows. The primary fragment with mje = 319 corresponds to mje = 44 (102 X 2) (14 X 4) 15. It therefore represents a fragment including the methyl terminus of the molecule (Formula 4), arising from cleavage of the bond between the third pair of methoxyl-substituted carbons, numbered from the methyl terminus. The fragments arising from cleavage between the first, second, fourth, and fifth pairs of methoxyla and b are characteristic.
+
+
+
+
+
=
44
+ 102y + 14b + 15
+
(102 X 4) (14 X 4) 15 = 523, respectively. All these fragments are represented in the spectrum except m/e 523. Therefore, the original compound contained four double bonds ( x = 4, Formula 2). The primary fragment with mje = 349 corresponds to ni/e = 59 (14 X 2) (102 X 2) 58. It therefore represents a fragment including the carboxyl end of the molecule (Formula 3) arising from cleavage of the bond between the third pair of methoxyl-substituted carbons, numbered from the carboxyl end. The fragments arising from cleavage between the first, second, fourth, and fifth pairs of methoxylsubstituted carbons would have rnje values of 145, 247, 451, and 553, respectively. All these fragments are represented
+
+
+
+
Figure 8. Mass spectrum of the derivative of 5,8,11,14,17eicosapentaenoicacid VOL. 40, NO. 12, OCTOBER 1 9 6 8
1843
in the spectrum except m/e 553, confirming the presence of four double bonds in the original compound. The values of a = 2 and b = 4, obtained from the q u a tions shown above, complete the assignment of the structure of the derivative as shown in Figure 7. This assignment of structure is not dependent on the presence of a molecular ion peak in the spectrum. Knowledge
Bi
acids other than the common straight chain acids with methylene-interrupted unsaturation will require a more complex trial and error method, but should be unambiguous.
Table I. Intensities of Principal Peaks in the Spectra of Derivatives of Polyunsaturated Fatty Linoleic a-Linolenic y -Linolenic Arachidonic of of of of mle base peak m/e base peak mle base peak mle base peak 201 8.1 201 5.7 159 53.9 145 24.9 169 2.2 169 2.5 127 31.7 113 5.4 137 5.6 137 4.1 95 15.0 81 1.6 277 6.1 319 4.8 421 217 13.4 1.4 10.4 287 5.4 389 100 245 0.8 185 35.7 255 6.7 7.8 213 357 0.6 153 181 12.9 223 15.6 325 2.0 149 8.9 293 1.9 261 2.2 229 2.3 197 5.2
z
Ai
of the gas chromatographic retention time of the original fatty acid methyl ester and of the polymethoxy derivative facilitates the assignment of the structure. The determination of structures for derivatives of fatty
z
z
z
303 271 239
5.4 30.4 6.4
261 229 197
13.8 38.9 29.9
247 215 183
4.3 13.9 12.2
175 143 111 79
13.8 73.2 7.3 2.1
217 185 153 121
16.7 95.8 4.7 1.8
A8
405 373 341 309
0.7 1.2 1.2 4.3
363 331 299
2.4 3.8 4.0
3.7 3.3 3.0 4.9 1.9 2.7 3.0 1.5 4.6
&
73 41
26.8 7.5
115 83
21.6 13.2
319 287 255 223 191 349 317 285 253 221 217 185 153 121
Ai
&
303 271 239
62.2 17.6
115 83
10.6 11.2
4.0
x=2 a = 6 b = 4
x = 3 a = 6 b = l
x = 3 a = 3 b=4
5.0
6.1 39.4 2.6 1.7
451 419 387 355 323 291 259 227
A4
1.2 0.8 0.5
2.6 1.7 2.1 1.5 6.3
115 83
15.1 5.0
x = 4 a = 2 b =4
Acids Eicosapentaenoic of mle base peak 145 25.4 113 6.2
z
481 449 417 385 353 321 289 257 225 247 215 183 151 379 347 315 283 251 349 317 285 253 221 277 245 213 181 149 451 419 387 355 323 291 259 227 195 175 143 111
0.6 0.5
0.2 1.4 1.7 1.3 1.2 1.3 1.9 2.8 8.7 12.7 2.1 1.6 1.1 1.0 3.0 2.5 1.2 2.3 2.3 3.9 5.2 2.2 4.2 5.2 6.2 3.4 1.3 0.9 0.7 2.5 1.5 2.3 1.6 5.8 3.4 5.8 35.1 6.3
553 521 489 457 425 393 361 329 297 73 41
Bs
0.4 0.2 0.2 0.6 0.9 0.8 0.5
1.1
1 .o
15.0 5.5
x = 5 a = 2 b = l 1844
ANALYTICAL CHEMISTRY
Table 11. Peak Matching of Fragments from 7-Linolenic Acid Derivative. Major Mlow Mobserved contributing fragment 159.1325 2 159.1021 159.1385 z (CsHi,03) A (CsHisOa) 223,1630 B-3x32 223.1334 223 223.1700 B-3x32 Y-3x32 (C13H1903) (C14H2302) C - 32 229 229.1803 229.1440 229.1413 C - 32 X - 32 (C12H2104) (ClaH2503) D - 32 185 185.1541 185.1177 185.1466 W - 32 D - 32 (CiiHziOz) (CIOH1103) 267 267.1960 267.1596 267.1559 E-3x32 E-3x32 V-3X32 (ClB"2703) (Ci5H2804) Reference compounds used were n-undecane (m/e 156;1878), n-dodecane (m/e 170.2034), n-pentadecane (m/e 212.2504), and n-octadecane (m/e 254.9973). Mass number 159
Mhigh
0
IV. Resolution of Ambiguities in Spectra with the PeakMatching Device. Certain peaks in several of the spectra could result either from cleavage of the bond between methoxyl-substituted carbon atoms, or from cleavage alpha- to a pair of methoxyl-substituted carbons. This problem is particularly acute in the case of the derivative of y-linolenic acid,
I
w
u-v 115
363
I
into a special inlet system in which a gallium or membrane inlet is used. A fast mass-marker is used for quick selection of the peaks to be matched. The oscilloscopic peak matching is accomplished by a direct readout resistor decade. With a resolution of 1500, the value of m / e can be determined with an accuracy of 10 parts per million, Because the sample must be introduced into the ion source
I
x
Y -z
+--)
217
in which each of the expected primary fragments arising from cleavage between methoxyl-substituted carbons (fragments A-F) has a counterpart with the same formal mje, which could arise from alpha-cleavage (fragments U-Z). These fragments do not have identical elemental compositions, however, but constitute (0-CH4) doublets, differing by 0.0364m. With a peak-matching device developed in this laboratory for use with the LKB 9000 single focusing mass spectrometer ( 1 9 , we were able to determine the values of rnje for each of these peaks sufficiently accurately to assign the elemental composition, and thus we were able to determine which member of the (0-CH4) doublet made the major contribution to each peak. With this device, the unknown peak and a reference peak are simultaneously displayed on an oscilloscope screen. The unknown and reference peaks must not differ by more than 10% of mje. The unknown sample (0.1-1 pg) is introduced by the direct probe, and the reference compound is introduced ( 1 5 ) R. Ryhage, 15th Annual Conference on Mass Spectrometry and Allied Topics ASTM Committees E-14, Denver, 1967.
261
319
159
uia the direct probe, the sample of 6,7,9,10,12,13-hexamethoxy methyl octadecanoate (derived from y-linolenic acid) was purified by chromatography on silicic acid prior to analysis. The results obtained are summarized in Table 11. In most cases the peak chosen for analysis was not the primary fragment (A-F, Formula 5 ) , but a secondary fragment arising from the loss of the elements of methanol, because the peak matching is more easily accomplished with peaks of higher intensity. With the exception of the peak at mje 159, which arises from cleavage alpha- to the final pair of rnethoxylsubstituted carbons, all peaks result from cleavage between methoxyl-substituted carbons. The relative intensities of the various primary fragments (A-F) in the spectra of the derivatives of CY- and y-linolenic acids are quite similar, with the exception of fragment A (mje 201 and 159, respectively) (see Figures 5 and 6 and Table 11). By this method, we have demonstrated that the primary contribution to the peak at m/e 159 comes from the fragment produced by cleavage between C Iand ~ CIS,and not from "fragment A." The spectra of the derivatives of CY- and y-linolenic acids therefore each contain a relatively intense peak (greater than 40% of the base peak) corresponding to cleavage alphaVOL. 40, NO. 12, OCTOBER 1968
* 1845
l
Figure 9. Mass spectrum of a mixture of derivatives of petroselenic, oleic, and vaccenic acid
to the final pair of methoxyl-substituted carbons (mje 117 and 159, respectively) and a much less intense peak corresponding to fragment A . In addition to the fragments arising from cleavage of bonds adjacent to the methoxyl-substituted carbon atoms, prominent peaks are present at m/e 71, 75, and 101. These peaks were examined with the peak-matching device to determine their elemental compositions. The peak at mje 71 has the composition C4H70(m/e 71.0497), experimental value 71.0496, reference-benzene. The peak at mje 75 has the composition C3H702(mje 75.0446), experimental value 75.0446, referencebenzene. The peak at mje 101 has the composition C~H902 (m/e 101.0603), experimental value 101.0604, referencetoluene. The peak at m/e 71 is present in all the spectra and we propose the structure H
I
C+ H2C-
/\
CH
The peaks at m/e 75 and 101 are present in the spectra of all those derivatives containing the grouping -CH-CHz-CH--CH-
I
I
I
OCHa OCH3
(Figures 3-8) and we therefore propose the following structures: H
I
C+
/\
m/e 75; 0
0
I
H
I
C+
/\
CH
I
OCH3 OCH, This structure for the ion at mje 75 in the spectra of o-methylated compounds has also been proposed by Grutzmacher and Winkler (16). (16) H. Grutzmacher and J. Winkler, International Mass Spectrometry Conference, Berlin, Sept. 25-29, 1967. 1846
ANALYTICAL CHEMISTRY
6
Will,
8
i
r
r
t
o
Figure 10. Resolution of the derivatives of petroselenic, oleic, and vaccenic acids with the accelerating voltage alternator
V. Determination of the Composition of a Mixture of MonoUnsaturated Fatty Acids. The gas chromatographic retention times for a number of the derivatives tested are shown in Table 111. The derivatives of various C18 monounsaturated fatty acids were not resolved on nonpolar (SE-30) or polar (EGSS-X) columns. The composition of a mixture of monounsaturated fatty acids of equal chain length must therefore be determined by interpretation of the mass spectrum of the mixture of derivatives produced. The mass spectrum of a mixture of the derivatives of petroselenic acid (CH A 6), oleic acid (CISA 9), and vaccinic acid (CISA 11) is shown in Figure 9. The fragments corresponding to A and B (cf. Figure 1) are: for petroselenic acid, m/e 159 (A) and 199 (B); for oleic acid, mje 201 (A) and 157 (B); for vaccenic acid m/e 229 (A) and 129 (B). The peak at m/e 197 occurs only in the spectrum of the vaccenic acid derivative, and corresponds to a loss of methanol from fragment A (229 - 32). The fragments with m/e = 327, 295, and 263 are common to the three derivatives, and represent M - 31, M - (31 32), and M - (31 2 X 32), respectively. Knowledge of the gas chromatographic retention time facilitates the interpretation of this otherwise complex spectrum. From this spectrum the composition of the mixture may be determined qualitatively, but the percentage composition may not be calculated. The intensity of a given peak in the mass spectrum depends on 1) the quantity of the derivative introduced into the ion source of the mass spectrometer, 2) the efficiency with which this derivative is ionized, and 3) the fraction of the total ion current caused by the particular fragment. One may use standards to determine the intensity of the various peaks relative to the quantity of derivative introduced into the ion source, under a given set of experimental conditions. Given these values for the various components of a mixture, one
+
CHI
and
I
h
1
CH3
m/e 101; HC-
a
+
OCHa
OCH3
z
Table 111. Gas Chromatographic Retention Times of Derivatives of Unsaturated Fatty Acids Equivalent carbon numbera Fatty acid Oleicb 20.4 Ricinoleic 21.3 Linoleic 22.2 y-Linolenic 23.9 a-Linolenic 24.5 Arachidonic 27.2 a Relative to saturated straight chain fatty acid methyl ester. The column was 1 SE-30on gas-chrom. P. b The derivatives of Cls monounsaturated fatty acids were not resolved.
may approximate the percentage composition of the mixture from its mass spectrum. However, another source of error arises from minor differences in the retention times of the various derivatives, so that the chromatographic peaks are not precisely superimposed. Thus, a mass spectrum taken at the apparent peak of the chromatographic effluent will not be quantitatively representative of the entire mixture. With an accelerating voltage alternator developed in this laboratory for use with the LKB 9000 instrument (17), we were able to determine the distributions of the various derivatives within the chromatographic peak. With this device, simultaneous recordings are made of the changing intensities of three selected m/e values during elution of the mixture from the chromatographic column. Peaks for the three ions are observed with a single collector, one oscillographic recorder, and at constant magnetic field strength, by rapid switching of accelerating voltage with a time actuated relay and voltage dividing circuit. A value of m / e was chosen for each derivative to satisfy the following criteria: 1) it must be a relatively intense peak in the spectrum; 2) it must have a very low intensity in the spectra of the other derivatives; 3) the three mje values must differ by less than 10% of mje. For this experiment we used for the petroselenic acid derivative, m/e 199 (fragment B ) ; for the oleic acid derivative, m/e 201 (fragment A ) ; and for the vaccenic acid derivative, m/e 197 (fragment A - 32).
(17) C. Sweeley, W. Elliott, I. Fries, and R. Ryhage, ANAL.CHEM., 38, 1549 (1966).
The plot of total uncorrected ion intensities us. time obtained with an equimolar mixture of these three derivatives is shown in Figure 10. Note that the order of emergence from the column is 6,7-dimethoxy methyl octadecanoate, 9,lOdimethoxy methyl octadecanoate, 11,12-dimethoxy methyl octadecanoate. The areas of the peaks were measured, and correction factors were calculated, such that area x correction factor = 100 for each curve. Mixtures with other molar compositions were prepared and the ratios of the derivatives in these mixtures were determined by the above described method. The areas of the three peaks were measured and multiplied by the appropriate correction factor. For a mixture of 6,7-dimethoxy methyl octadecanoate: 9,lO-dimethoxy methyl octadecanoate: 11,12-dimethoxy methyl octadecanoate (1 :2 :3)) the experimentally derived ratio was 1:2.0:2.7; for a 3:2:1 mixture, the ratio 2.7:1.8:1 was found; for a 1:3:1 mixture, 1.1:2.8:1. Thus, the percentage composition of a mixture of monounsaturated fatty acids of equal chain length may be determined to at least an accuracy of &lo%. ACKNOWLEDGMENT
We are grateful to Mrs. Britta Johansson and Miss Yvonne Jansson for expert technical assistance. RECEIVED for review March 4, 1968. Accepted June 27, 1968. This work was supported by The Medical, Natural and Technical Science Research Councils of Sweden. Walter Niehaus was the recipient of a postdoctoral fellowship from the National Heart Institute, USPHS.
Thermodynamically Based Gas Chromatographic Retention Index for Organic Molecules’ Using Salt-Modified Aluminas and Porous Silica Beads Donald T. Sawyer and David J. Brookman Department of Chemistry, Uniaersity of California, Riverside, Calif. 92502
A retention index has been developed for salt-modified aluminas and porous silica beads which is based on the evaluation of the enthalpies and entropies of adsorption for a series of hydrocarbons and substituted aromatic hydrocarbons. A direct relationship between the logarithm of the retention volume and the free energy of adsorption permits the prediction of the retention volume at any temperature within the usable range of the index system. By assuming that the various functional groups of the molecule contribute in an additive fashion to the thermodynamic quantities the retention volume of the molecule can be predicted from its formula. From an evaluation of the retention parameters for a group of salt-modified columns, prediction of the ideal adsorbent and column conditions for a given separation is possible. Examples of predicted separations of difficult mixtures are given. THE UTILITY of salt-modified aluminas for gas chromatographic separations of hydrocarbons was first discussed by Scott (1). This early work was developed in a more complete (1) C. G. Scott, “Gas Chromatography 1962,” M. van Swaay,
Ed., Butterworths, Washington, 1962, p 36.
discussion in 1964 (2) which demonstrated that a high degree of selectivity is possible for the separation of isomeric mixtures. Recent studies (3, 4) have established that the interaction between a sorbate molecule and a salt-modified alumina is a combination of nonspecific and specific contributions. The latter include effects due to the pi character of the sample molecule and its geometric configuration. For aromatic hydrocarbons, substituent groups affect the gas-solid interaction and for olefins the cis isomer is retained more strongly than the trans isomer. In the previous study (3), plots of the retention volume logarithm as. the boiling point for the members of a homologous series of sorbate molecules have been shown to give smooth curves that approach linearity. The symmetry of the elution peaks provides additional support for the conclusion that salt-modified aluminas provide a surface which allows (2) C . G. Scott and C. S. G. Phillips, “Gas Chromatography 1964,” A. Goldup, Ed., Institute of Petroleum, London, 1965, p 266. (3) D. J. Brookman and D. T. Sawyer, ANAL.CHEM., 40,106 (1968). (4) G. L. Hargrove and D. T. Sawyer, ibid., p 409. VOL. 40, NO. 12, OCTOBER 1968
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