To
I=
(1820 i 4.50)
+ (11.25 f 0.7) X
i2p
(18)
From the intercept of this line, a true temperature of peak evaporation Tt = 1820 OK could be derived. From the slope and the value of 1 = 15 X 10-2 cm, a value of IJ = 15 cm/sec could be computed. This low value of velocity could explain the increased analytical sensitivity of the flameless in respect to the flame spectroscopy. As alternative explanation of the increasingly higher temperature of the peak, a kinetic effect could be claimed. Actually a plot according to Equation 9 fitted nicely a straight line giving AG* of the same order of magnitude of the sum A H f u s i o n + AHvaporization. The real situation probably lies somewhere in between these two extreme cases, but in order to separate the two effects it
would be necessary: (1) to have an independent measurement of the velocity of the evaporating atoms a t the different thermal conditions, and (2) a more linear temperature programmer since in kinetic investigations a variation of &lo%of the heating rate is no longer satisfactory. Both these problems are currently under study in this laboratory.
ACKNOWLEDGMENT Thanks are due to V. Sacchetti and S. Giodice of the mechanical shop for cooperative help in the design and construction of the atomizer. Received for review November 7, 1972. Accepted February 15, 1973. Partial financial support from Centro di Chimica Analitica Strumentale is acknowledged.
Analysis of Triglyceride Mixtures by Gas Chromatography- Mass Spectrometry fakeshi Murata and Seiji Takahashi Analytical Application Laboratory, Kyoto Laboratory, Shimadzu Seisakusho Ltd., Nakagyo-ku, Kyoto, Japan
A combined system of a gas chromatograph and a mass spectrometer permits rapid analysis of triglyceride samples. A sample is first separated on the carbon number basis by a gas chromatograph and the constituent fatty acids are identified by a mass spectrometer. Thus the fatty acid composition and the molecular weight distribution were determined. The fatty acid composition of 14 groups of 78 types of triglycerides, ranging from 28 to 54 in carbon number were determined and the results compared with those of preparative gas chromatography. They agree quite well in general, except for c6-c1O-c12 of (228, C ~ - C ~ - CofI ~C ~ Oand , c14-c14-c16 of C44 triglyceride. This method was applied to the analysis of the triglycerides of cows' milk fat, bovine fat, rat liver, and rat blood, with carbon numbers of 46, 48, 50, 52, 54, and 56, and their fatty acid composition determined. We also determined the double bonds of the fatty acids combined with the glycerine from the molecular weight distribution. We did not proceed to determine the potential isomers of the trigIycerides-Cl6-Cl8-Cl4 YS. c18-c16-c14 YS. CI8CI4-Cl6-or to discriminate the isologs (C18:o-C18:oc18:oYS. c18:o-c18:1-c18:1 YS. c18:1-c18:1-c18:2).
With the recent remarkable development of gas chromatography, there have been many studies made on the analysis of lipids. The use of this technique has made it possible to separate triglycerides on the total fatty acids or on the carbon number basis ( I ) . There are also reports (2-4) that coconut oil, which is a (1) A. Kuksis, "Gas Chromatography of Neutral Glycerides Lipid Chromatographic Analysis," Vol. 1, Marcel Dekker, New York, N.Y., 1900. (2) A. Bomer and J. Baumann, Z. Unters. Nahr. Gebrau. chsgegenstaende, 40, 97 (1920). (3) G. Collin and T. P. Hilditch, J. SOC. Chem. lnd., London, Trans., 47, 261 (1928). (4) A. Dale and M. Meara, J. Sci. FoodAgr., 6, 162 (1955). 1816
mixture of triglycerides, was fractionated into nine triglycerides through fractional crystallization, and the pattern of the fatty acid composition was obtained. There are also reports (5-7) on the determination of the fatty acid composition of coconut oil or some natural fats by chromatography, but the results are not very satisfactory. Samples like triglyceride mixtures must be gas chromatographed a t a very high column temperature and with small sample sizes, which makes it difficult to collect the fractions. Bugaut et al. have succeeded in determining the fatty acid composition of 79 coconut oil samples having triglycerides of 13 groups, ranging between 28 and 52 in carbon number (8, 9). But their method cannot be applied to the analysis of triglycerides of larger carbon numbers, which have higher boiling points and complicated structures. Employing mass spectrometry, Barber et al. ( I O ) studied [M - RC02]+ ion, [RCO + 74]+ ion, [RCO + 128]+ ion, and RCO+ ion, which are molecular ions ( M + ) without acyloxy groups, using tristearin and 1-myristo-2-steara3-palmitin as samples. Lauer et al. prepared triglycerides labeled with deuterium and made a very detailed study of many mass fragments to obtain information on the structures using single focus and double focus mass spectrometers ( I I ) . As for triglyceride mixture samples, Hites (12) has only been successful in determining the number of double (5) A. Kuksis and M. T. McCasthy, Can. J. Biochem. Physioi., 40, 679 (1962). (6) P. Augustin. Oleagineux, 22, 99 (1967). (7) W. C. Breckenridge and A. Kuksis, Lipids, 3,291 (1968). (8) M . Bugaut and J. Bezard, J. Chromatogr. Sci., 8, 380 (1970). (9) J. Bezard, M. Bugaut, and G. Clement, J. Amer. Oil Chem. SOC., 48, 134 (1971). (10) M . Barber, T. 0 . Merren, and W. Kelly, Tetrahedron Lett., 18, 1063 (1964). (11) W. M. Lauer, A. J. Aasen, G. Graff, and R. T. Hoiman, Lipids, 5(11), 861 (1970). (12) Ronald A. Hites, Anal. Chem., 42, 1736 (1970).
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, N O . 11, SEPTEMBER 1973
i /
10
s-
20 190
Figure 1. Chromatogram of
42
30
2 10
240
300
320
min
.c
coconut oil detected by a total ion collector
(The carbon numbers are written on the peaks.) Conditions: 0.35 programmed temperature
X
3 mm i.d. glass column packed with 1% OV-1; 140-310 "C (at 4 "C/min)
bonds from the parent ions of 48, 50, 52, and 54 in carbon number, about kokum butter and cocoa butter samples, using a mass spectrometer and an IBM 1130. We tried to analyze a triglyceride mixture contained in mconut oil, cows' milk fat, head fat, rat liver, and rat blood. We used a combined system of a gas chromatograph and a mass spectrometer and succeeded in getting knformation on the fatty acid distribution through calcula;ion of patent ions and mass fragments such as [M RC02]+ ion and RCO+ ion and were able to determine ;he number of double bonds. The following is a report of our experiment.
Table I. Molecular Weight of the Triglyceride Ranging between 28 and 56 in Carbon Number Carbon No. MWn 56 54 52 50 48 46 44 42 40 38 36 34 32 30 28 26
EXPERIMENTAL Materials. Tristearin (TS), tripalmitin (TP), and trilaurin
TL) were purchased from Allied Science Laboratory Inc. The nilk fat had a purity better than 98%. The bovine fat was puriied to be 95% pure through solvent extraction. The rat liver and he rat blood were offered by the surgical laboratory of Kyoto Jniversity. The neutral lipid was isolated by the Folch Method. rhe samples were stored at a temperature below 5 "C. Instrumentation. The equipment used was: GC-MS-Shinadzu LKB 9000 gas chromatograph-mass spectrometer com)ined system, and GC-Shimadzu GC-4BM gas chromatograph. The gas chromatographic conditions were as follows. The colimn was 0.35 m X 3 mm i.d., glass coil with 1% OV-1 on Chronosorb W, 80-100 mesh. The chromatograph was programmed rom 200 to 330 "C a t 4 "C/min and kept isothermal a t 330 "C ntil the last peak was recorded. The sample injection port temierature was maintained at 350 "C. The carrier gas was helium, 5 ml/min. A Ryhage type separator was used and set a t 350 "C. i total ion collector was used as the detector for GC-MS. The mass spectrometric conditions were as follows. The ion ource temperature was held at 330 "C during the GC-MS runs. 'he mass spectra were all obtained at 20 eV of electron energy, .5 kV of accelerating voltage, and 60 pA of trap current. The can speed was 7. All samples were diluted with CHC13 to about 0%. Fatty acids were chromatographed on a 25% DEGS column Zhromosorb W, 80-100 mesh).
RESULT AND DISCUSSION Fatty Acid Determination of Triglycerides on Carbon (umber Basis. Figure 1 shows a chromatogram of cocout oil, detection being done by a total ion collector. The pikes on the peaks show sampling for mass spectrometry. Ryhage ( I 3 ) , Barber (IO), and Lauer (11) studied their lass spectra by directly introducing pure TL, TP,and TS nto a mass spectrometer. We selected the [M - RC02]+, 13) R.
40
270
Ryhage and E. Stenhagen, J . LipidFfes., 1.11361 (1960).
MW = 134
+ n X 14.
918 890 862 834 806 778 750 722 694 666 638 61 0 582 554 526 498
+
[RCO 74]+, [RCO + 128]+, and RCO+ ions from the mass fragments and used them for the determination of the fatty acids. Table I shows the molecular weights of the triglyceride ranging between 28 and 56 in carbon number. The carbon numbers were determined from the retention times of the pure triglycerides or from the parent ions of the mass spectrum. The determination of the carbon number or the molecular weights is very important, because without knowing those of the parent ion M + , it is impossible to determine the RCO2 in [M - RC02]+. Take the m / e 439 for example. If the carbon number of the triglyceride is known to be 34, that is, 610 in molecular weight, the m / e of RCO2 must be 171 because 610 - 171 = 439. This means that C ~ fatty O acid exists in the Cz4 TG. If the molecular weight is 638, that is, 36 in carbon number, the m / e of the RC02 must be 199 because 638 199 = 439. This means that Cl2 fatty acid exists in the C36 TG. Thus, if a different supposition is made about the molecular ion, a fatty acid of different carbon number is presumed to exist. Determination of the Fatty Acid Composition of C 3 4 TG. The molecular weight of C34 is 610 and the [M RC02]+ ions appear a t m / e 495, 467, 439, 411, 383, 355, and 327. From these it can be deduced that RC02 ions are
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
1817
+
Table II. Values of RCOf, [RCO 7 4 ] + , and [RCO 128]+ for Various Carbon Numbers Fatty acids
RCO
RCO
71 99 127 155 183 21 1 239 267 265 263 295 293 29 1 289 28 7 323 32 1 31 9 31 7 315 313
c4
CS c6 c10
c12 c14
c16
Cl8 0 Cl8 1 C182 c20 0
c20 1 c20 2 c20 3
c20 4 c22 0 c22 1
c22 2 c22 3 c22 4
c22 5
+ 74
145 173 201 229 257 285 313 341 339 337 369 367 365 363 361 397 395 393 39 1 389 387
RCO
+
+ 128
199 227 255 283 31 1 339 367 395 393 39 1 423 421 41 9 41 7 41 5 451 449 447 445 443 441
115, 143, 171, 199, 227, 255, and 283. Therefore, we concluded that there existed fatty acids of 6, 8, 10, 12, 14, 16, and 18 in carbon number. Thus, fatty acid composition can be determined from the [M - RC02]+, but determination from a single fragment is not very reliable because many fragments of the hydrocarbon chain of fatty acid appear on the mass spectra. Therefore, we also used RCO, RCO + 128, and RCO 74 fragments in our study. The RCO + 128 of the C34 TG appeared a t m l e 99, 127, 155, 183, 211, 239, and 267. From all of these data, it can be deduced that the fatty acid compositions of the C34 TG are c 6-ClO-Cl8, c 6-ClZ-cl6, c6-c 14-Cl41 cS-CS-cl8, c8-c1O-c16, cS-Cl2-cl4, C~O-CIO-CI~, and clo-c12-c12. Table I1 shows the values of [RCO 128]+, [RCO + 74]+, and RCO+ for various carbon numbers. Table I11 shows the fatty acid composition of triglyceride groups ranging between 28 and 54 of coconut oil. The values in the a column were obtained, by Bezard e t al. (9), through fractionation, hydrolysis, esterification of fatty acids into butyl esters, and gas chromatography. The values in the b column were directly obtained through calculation from the values of RCO+ ions. In the case of a, which utilizes esterification of fatty acids, the fatty acid composition can be simply obtained by multiplying correction factors of the chain lengths to the peak areas. But in the case of b, not all of the mass fragments are useful for quantitative purposes. It is necessary to select the quantitative fragments, and, sometimes, to use the mean values of more than one fragment. Bezard compared the results of their GC fractionation with the random distribution of triglyceride groups or triglyceride types obtained by Kuksis et al. calculated from the Bailey method. Comparison of Our Results with Those Obtained by t h e Bezard M e t h o d . As already reported by Lauer ( I I ) , the - RC02]+ ion is convenient to identify existing fatty acids but it is not very useful to quantitate them. As for 128, and RCO ions, the former two the RCO 74, RCO ions have the same characteristics. Therefore, we compared the RCO+ and the [RCO + 128]+ ions. Concerning the carbon numbers 28 and 32, the fatty acid carbon number 6 is recorded higher than the RCO+ ion. With carbon numbers 30 and 36, the RCO 128 is recorded high in the short chain region but it has characteristics similar to
+
+
+
+
+
1818
RCO+. As for the c38, c 4 0 , c 4 2 , c46, and c48, the c16 and CIS fatty acids are recorded lower because the Clz fatty acid is recorded higher because of the higher response of the RCO + 128. In short, RCO+ and [RCO + 128]+ ions provide almost the same results, though there are minute differences. As reported by Barber (IO), the ion abundance of the RCO+ or [RCO 128]+ does not differ with the positions (c14,c16, or CIS) in l-myristo-2-steara-3-palmitin,or with the lengths of the chains. In the region between c6 and CZO,the length of the carbon chain has some relation with the intensity of the RCO+ ion, which we neglected in our calculations. The influence of the background current was proved negligible in our test, using the background from the OV-1 column and the fragments of TL, TP, and TS. This can be well understood from the comparison of the RCO+ method and the data of Bezard. The C6-cZO fatty acids of TG were plotted based on the number of TG on the abscissa and the fatty acid content on the ordinate. This showed us the patterns of c6, CS, and Clo are quite similar, being a little higher in the GC-MS method. Clo is almost the same as the c6, CS, and Clo in the GC fractionation method, but this may be mainly due to the high volatility of these lower fatty acids during extraction and butyl esterification. There is a rather large difference in the response of C12 fatty acid which is the major component of coconut oil. Especially concerning the T G of 46, 48, and 50 in carbon number, the GC-MS method gives values more than twice as large. c14 fatty acid gives values about four times larger than for TG of 50 in carbon number. The values of Clz and c14 change largely a t carbon number 50. and ClS give patterns similar to c6, Cs, and Clo. The lower values of GC-MS in C44, (346, and C48 TG may be due to the higher values of C12 and c14. (Since the values in Table 111 are in percentages, if one value becomes larger, another naturally becomes small.) Since GC-MS analysis was done up to Cs4, much C20 fatty acid appeared. Table 111 shows the total fatty acids of coconut oil. The three sets of results are satisfactorily similar. Figures 1-4 show chromatograms, using TIC of the GC-MS system. The present chromatographic technique cannot completely separate these triglycerides. This may cause error in the identification after collection from gas chromatography, but in actual GC-MS operation, the sampling time into the mass spectrometer is 4 sec for c28-c44 T G and 5 sec for c46-Cb4 TG. When the total gas chromatographic time is 2-3 min, therefore, there is only a small possibility that the components of the next peaks will be contained in the component to be introduced into the mass spectrometer. The mass spectra of C34 TG of coconut oil, C ~ O(252, , and C54 T G of rat blood, and C54 and c56 TG of rat liver are shown in Figures 5-10. Determination of Saturation of Fatty Acids for Each Carbon Number of Triglycerides. Unlike gas chromatographic separation of saturated and unsaturated fatty acids, the separation by the GC-MS system is very simple; because of the mass difference of 2 amu among ClS:O, C l s : ~ ,and the RCO+ appears a t m l e 267 only when C l s : ~exists and a t m l e 265 and 263 when C18:1 and Cia::! exist. The intensities of these fragments provide quantitative information. Figures 11 and 12 show the concentration ratio of C18:0, and Cls:2 for various carbon numbers of TG of cows’ milk fat, bovine fat, rat blood, and rat liver (Cl8:O = 100). This datum does not , and C18:2 show the concentrations of T G in C l s : ~ &:I, fatty acids but the ratio of three Cls carbon number fatty acids in each peak. The particular patterns for different
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
+
38 34
I
50
IO
180
54
230
25
20
IS
200
'*
h
42
260
290
rain
320
S
'C
Figure 2. Chromatogram of cow milk fat triglyceride detected by a total ion collector The carbon numbers are written on the peaks
Table Ill. Fatty Acid Composition of Triglyceride Groups 28 to 5 4 of Coconut Oil Triglycerides Triglyceride group Fattyacids 6:O
a b
8: 0
a b
10:0
a b
12:O
a b
14:O
a b
16:O
a
28
30
32
34
36
38
40
9.7 5.0 36.8 43.0 12.5 18.0 28.7 31.0 5.3 3.0 4.3
10.2 26.0 19.2 27.0 20.1 19.0 41.3 27.0 2.4 1.0 4.1
3.2 15.0 26.5 42.0 5.9 10.0 54.3 31.0 4.4 2.0 2.3
1.9 10.0 18.6 31.0 12.6 15.0 44.4 35.0 16.5 8.0 3.9 1.0 1.3 1.0 0.1
1.1 2.0 8.5 15.0 9.7 14.0 58.9 60.0 11.4 9.0 8.2 2.0 1.2 2.0 0.1
0.5 1.0 7.2 20.0 5.9 9.0 51.0 53.0 23.0 13.0 6.1 3.0 3.5 3.0 0.1
0.5 1.0 3.3 10.0 6.0 11.0 44.2 53.0 23.8 16.0 14.3 5.0 4.8 5.0 0.3
b
18:O
a
2.6
1.7
2.1
b
20:O
a
0.2
0.2
0.2
42
Total triglycerides 44
46
48
50
52
54
a
b
3.9
C
1.3 6.0
2.2 1.0 1.8 6.0 41.7 58.0 17.4 14.0 18.3 6.0 11.0 6.00.3
b
3.0 1.0 2.0 7.0 22.8 60.0 25.5 19.0 20.2 6.0 19.0 6.0 0.8 6.0
1.6
0.9
2.5
4.5
11.0
12.0 14.0
3.0 10.0 23.5 60.0 15.2 14.0 28.7 10.0 22.1 8.0 0.8 4.0
1.9 2.0 15.2 50.0 14.1 15.0 26.1 17.0 33.1 6.0 1.0 1.0
0.9
2.0
8.1
8.0 8.0
5.3 5.0 10.8 52.0 42.0 22.0 29.8 22.0 1.2 2.0
8.4 6.0 7.0 32.0 34.4 32.0 28.5 32.0 2.5 5.0
34.0
48.2 40.0
13.5
14.6 11.0
16.6 37.0
6.9 8.0
12.3 52.0
7.9 10.0
0.6 11.0
0.1 3.0
*
a As determined by the fatty acid composition of fractionated triglycerides. As determined by the fatty acid composition by means of the GC-MS combined system. As determined by analytical gas chromatographic separation of total fatty acids of coconut oil.
samples may be interesting. The [M - RC02]+ ion provides interesting datum. The M+ ion of c18:O-c6-c16 and C18:1-c&16 (40 in carbon number) appears at m l e 694 ] [M - C18:1] both appear a t m / e and 692. [M - C l ~ : oand 411 and the saturated and the unsaturated forms cannot be discriminated. But in the case of [M - c6]+ or [M c16]+, a doublet appears at m l e 411 and 399, which shows that c18:o-c6-c16 and c18:1-c6-c16 T G exist. M + Of CIS:O-C~-CI~:O, C I ~ : O - C ~ - C1,I ~ and : C~S/I-C~-CIS:Iap-
pear at m / e 694,692, and 690, [M - Cls:o]+ and [M - CIS:I] give a doublet a t m l e 321 and 325, and [M - C4]+ gives a triplet at mle 601,605, and 603. Thus, it is possible to detect unsaturated T G but quantitation is difficult to determine because of overlapped peaks. Triglyceride Type Composition. A triglyceride type is defined by its three constitutive fatty acids. The total of the carbon numbers of the constituting fatty acids is
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 11, SEPTEMBER 1973
1819
Table I V . Comparison of the Distribution of Triglyceride Groups and Triglyceride Types in Coconut Oil 28
30
32
34
36
a
b
6- 6-16 6- 8-14 6-1 0-1 2 8- 8-12 8-1 0-1 0
8 10 30 50 2
2 6 63 29
6- 6-18 6- 8-16 6-10-14 6-1 2-1 2 8- 8-14 8-1 0-1 2 10-1 0-1 0
3 3 37 2 43 4
6- 8-18 6-10-16 6-12-14 8- 8-16 8-10-14 8-12-12 10-10-12
6 2 7 2 4 75 4
1 1 1 85 12
6-10-18 6-12-16 6-14-14 8- 8-18 8-10-16 8-12-14 10-10-14 10-12-12
2 7 2 4 5 48 2 30
1 1 10 1 1 48 5 33
6-12-18 6-14-16
2 2
1 1
36
2 11 21 45 19 2
38
40
42
44
8-10-18 8-1 2-1 6 8-14-14 10-1 0-1 6 10-12-1 4 12-12-12
3 12 12 10 7 52
1 8 12 3 12 62
8-12-18 8-14-16 10-1 0-1 8 10-1 2-1 6 10-14-14 12-1 2-1 4
15 7 3 10 1 64
2 3 1 10 11 73
8-14-18 8-16-16 12-12-18 10-14-16 12-12-16 12-14-14
8 2 14 4 40 32
7 8 15 16 30 34
8-16-18 10-14-18 10-16-16 12-12-18 12-14-16 14-14-14
6 3 2 44 43 2
4 6 54 25 11
8- 8-18 10-14-20 10-16-18 12-12-20 12-14-18
10
'..
44 46
48
3 6 7 37
7 " '
51
12-16-16 14-14-16 8-18-20 10-1 6-20 10-18-18 10-1 4-20 12-1 6-1 8 14-14-18 14-1 6-1 6
20 2 2
18 29
8
9 4 66 9 10
... ... 2
... 60 15 15
8-20-20 10-1 8-20 12-1 6-20 12-18-18 14-14-20 14-16-18 16-16-16
2 2 2 40 2 40 12
1 1 60 4 32 10
50
10-20-20 12-18-20 14-16-20 14-18-18 16-16-18
2 10 10 20 58
2 7 12 26 53
52
12-20-20 14-18-20 16-16-20 16-18-18
2 8 30 60
2 10 18 70
54
14-20-20 16-1 8-20 18-1 8-1 8
, '
5 16 79
determined by Bezard et a/. (1) As determined by a GC-MS combined system. (2) As determined according to the method reported in Bezard et a/. Ail groups required simplifying assumptions to complete the calcuiations. a As
52
Y
LI S
IO
3 30
IL
min
.c
I
I
210
D 140
270
IS
100
310
r.0
n
I mhn
T
Figure 3. Chromatogram of rat blood triglyceride detected by a total ion collector
Figure 4. Chromatogram of rat liver triglyceride detected by a total ion collector
The carbon numbers are written on the peaks
The carbon numbers are written on the peaks
1820
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
COCONUT OIL c34 183
t
"= tt
127
I
Figure 5. Mass spectrum of
C34 triglyceride
of coconut oil
+ 128]+, [RCO + 74]+, and RCO+ c6 + 128 227 C6 + 74 C6 + 128 255 C6 + 74 283 ClO + 74 C i o + 128 C12 + 128 31 1 c,2 + 74 C14 + 128 339 + 74 C16 + 128 367 + 74 CIS + 128 395 c,* + 74
Figures indicated are the values of [M-RC02]+, [RCO M-Cs M-CB M-Cio MC12 M414 M-C~K M-Cie
49 5 467 439 412 383 355 327
c14
c16
173 20 1 229 257 285 313 341
99 127 155 183 21 1 239 267
R a t B l o o d $so
1M
t
* t I
a
t
,
313
1 263
I
551
367
I
Rat Blood C S 2
100
313 263
Bo
239
Figure 7. The mass spectrum of C5*TG of rat blood
Rat B l o o d C 5 4
iw
263 r
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
1821
Figure 9. The mass spectrum of
C54
TG of rat liver
Rat Liver CS6
x10
-/
I
i ,L 900
Bx,
Figure 10. The mass spectrum of
c56
TG of rat liver
,
= * I
Table V. Comparison of Triglycerides Determined by the GC-MS Combined System 400
400 Cow Milk Fat Oil
42
44
46
48
Carbon Number
50
52
12-1 6-20 12-18-1 a 14-14-20 14-1 6-1 a 16-1 6-1 6
3 42 3 27 29
...
...
60 20
20 58
50
14-1 6-20 14-18-1a 16-1 6-1 a
3 44 53
9 29 62
13 4 a3
10 20 70
52
14-18-20 16-1 6-20 16-18-1 a
3 13 a4
5 5 90
3 6 91
1 15 a4
54
14-20-20 16-1 8-20 18-18-1 a
3 11 86
...
10 30 60
2 6 92
Carbon Number
56
Figure 12. Ratio of C l s : ~ , Cle:l and Cls:? for various carbon numbers of TG (rat blood, rat liver) 1822
TG
30 24 46
4a
54
Figure 11. Ratio of CIS:^, C I ~ : and ~ , C18:2 for various carbon numbers of TG (cow milk fat oil, head oil)
Rat blood
TG
27 31 42
100
40
Rat liver
TG
10-18-1 a i2-16-ia 14-1 6-1 6
200
38
Head Oil
fatTG
46
300
200
Cow milk
16-18-22 16-20-20 18-18-20
... 20
6 94
.
I
.
22
7 14 79
equal to the carbon number of the triglyceride. The distribution of the triglyceride type in each group was determined from the fatty acid composition of the groups as follows: three fatty acids out of those found in each triglyceride group in which the sum of the carbon atoms corresponds to the carbon number of the group were combined in every possible way. We conducted our calculations on a n assumption that all the fatty acids are saturated. (The degree of saturation could be deduced from the [M - RC02]+ or RCO+, as described before.) Table IV shows the triglyceride distribution calculated from the concentration ratio in Table 111 through the Be-
* ANALYTICAL C H E M I S T R Y , VOL. 45, NO. l l , SEPTEMBER 1973
Table V I . Double Bonds of Triglyceride Compositions for Cow Milk Fat, Bovine Fat, Rat Blood, and Rat Liver Cow milk Rat Carbon Double No.
bonds
Mol Wi
50 50 50 50 50 52 52
4 3 2 1 0 6 5 4 3 2 1
826 828 830 832 834 850 852 854 856 a58 860 862 872 874 876 878 880 882 884 886 888 890 900 902 904 906 908 91 0
52 52
52 52 52 54 54 54 54 54 54 54 54 54 54 56 56 56 56 56 56
0 9 8 7 6 5 4 3
2 1 0 5 4 3
2 1 0
fat
14 32 54
23 45 32
6 17 30 33 14
Bovine fat Rat blood
... 50 50
13 54 33
15 55 40
27 50 23
50 33 17
liver 20 41 32
7
13 39 30 8
8 17 33 29 9
9 12 19 31
4
4
25
12 19
27 17 15
10
zard method and GC-MS method. In the case of the Bezard fractionation method, C54 could not be measured because of its small quantity and high boiling point. But by the GC-MS method this can be easily measured-it provided data up to c56 in rat liver sample. These data generally agree quite well with those of the fractionation method.
The only great differences are the values for Cs-Clo-Clz ~ c30 T G (22.5 times), and of CZST G (20%), C S - C S - C ~of c14-c14-c16 of c44 T G (18.5 times). Since it was proved that the GC-MS method is useful for the study of triglyceride mixtures, we proceeded in the analysis of more complicated samples which require a higher column temperature, such as triglycerides in cows’ milk fat, bovine fat, rat liver, rat blood, etc. The results are shown in Table V. As for the positions of fatty acid combination of glycerine, Barber (10) and Lauer ( 2 1 ) reported that [M RCO&Hz]+ ion formed from positions 1 and 3 is useful. But this method is useful for only one type of TG and is not useful in the case where many TG’s of the same carbon number exists. We described the method to determine the degree of saturation of fatty acids in the former chapter. But the number of double bonds can be determined from the parent ions. Hites (12) found that the degree of saturation could be directly calculated from the M+ ions. The Hites method differs from the GC-MS method in the manner of measurement. In the Hites method, the TG mixture is directly introduced into a mass spectrometer and the peaks that do not overlap those of the M + ion are used for calculation. On the other hand, in the GC-MS method, since the TG mixture is fractionated by GC, the M + ion is not overlapped by the other mass fragments. Table VI shows the result of the GC-MS method. A comparison of Tables V and VI suggests a possibility of further extension of the application of this method. Table V shows, for example, that C54 TG of the rat blood Z Oof, c16-C18-c20, and 92% contains 2% of C ~ ~ - C ~ O - C6% of Cls-Cls-Cl~. Table VI shows, on the other hand, that the fatty acids of the C54 T G of the rat blood have two or more, up to seven, double bonds. Received for review November 6, 1972. Accepted February 12, 1973. Presented in part at the International Congress on Analytical Chemistry, 1972, Kyoto, Japan.
Simple Direct Combination of Gas Chromatography and Vapor Phase Infrared Spectrometry J. E. Crooks, D. L. Gerrard, and W. F. Maddams Epsom Division, Research & Development Department, B P Chemicals International Ltd., Great Burgh, Epsom, Surrey, England
A combined gas chromatography/infrared spectrometry system is described. The components of a sample are separated by gas chromatography and are passed separately into a heated multireflection gas cell. A spectrum is obtained for each component, using conventional scanning conditions, and while the spectrophotometer is running, the flow of carrier gas through the column is stopped. When the spectrum of a particular component has been obtained, the carrier gas flow is resumed and the next component passes into the cell. Spectra may be obtained for as’ many as five components in one sample without seriously impairing the column resolution. Useful
spectra are obtained from 100-pg quantities of most organic compounds which, for a total sample size of 10 pl, permits examination of components down to the 1% level. The system is readily assembled and the constituent gas chromatograph and infrared spectrometer may be used for other analytical work.
Although the combination of gas chromatography and high resolution fast scanning mass spectrometry has proved a very powerful weapon for the characterization of the components of rather complex mixtures of organic
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
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