Computer Prediction of Triacylglycerol Composition of Vegetable Oils by HRGC

N. R. Antoniosi Filho / O. L. M e n d e s / F. M. Lan~.as* Instituto de Qu~mica de S~o Carlos, Universidade de S'~o Paulo, 13560-970, $5o Carlos (SP), Brasil

Key Words
bIigh resolution gas chromatography Lipid analysis Triacylglycerides Vegetable oils Computer prediction of composition

The major papers using these hypotheses were based on the determination of the triacylglycerol composition according a simple separation of the triacylglycerides into groups with the same number of unsaturated fatty acids in the glycerol moiety [5, 9, 10]. However, even with the development of more efficient separation methods, such as capillary GC, the identification of the triacylglycerides present in a lipid sample, such as a vegetable oil, has been a difficult process [11,

Summary
A simple computer method for predicting the triacylglycerol composition of vegetable oils from the falty acid COmposition is described. The results are employed to Identify the triacylglycerides present in vegetable oils SUch as peanut, olive, bacaba and patua pulp oils analyzed by H R G C on non-polar and polarizable Columns.

12].
In non-polar capillary columns, qualitative analysis is simplified, because the triacylglycerides are separated in groups having the same number of carbon atoms (CN separation). The high selectivity of polarizable stationary phases offers a more refined separation of the triacylglycerides because, in these phases, separation occurs according to the polarity of the fatty acids. Thus, on a polarizable column, the separation of triacylglicerides such as P O O and PLS (Table I), which are triacy]glycerides with same carbon number and degree of unsaturation but with different polarity it can TaMe I. Representation of FAMEs FAME Myristic Pentadecanoic Palmitic Palmitoleic c/s-ll-Hexadecenoic Margaric c/s-9-Heptadecenoic Stearic Oleic Vaccenic Linoleic Linolenic Arachidic Gadoleic Behenie Erucic Lignoceric Symbol M Pt* P Po Hx* Mg Hp* S O V L Ln A Ga Be Er Lg Shothand designation C14 : 0 C15 : 0 C16 : 0 C16 : 1A 9c C16 : 1A llc C17 : 0 C17 : 1A 9c C18 : 0 C18 : 1A 9c C18 : 1A lle C18 : 2A 9c, 12c C18 : 3A 9e, 12c, 15e C20 : 0 C20 : 1A 9e C22 : 0 C22 : IA 13c C'2,1 : 0

Introduction
The development of non-polar and polarizable stationary phases with high thermal stability has been rclerrcd to as high temperature capillary gas chromatograplly (I-IT-CGC), one of the most widely employed analytiCal methods for the determination of the triacylglycerol COmposition of fats and oils [1, 2]. blaving overcoming the difficulties in relation to the thermal stability of the stationary phases, there still remains the need to overcome the difficulties in identification of the triacylglycerides present in these COmplex samples. In spite of this, ever since 1860, some approachcs based on the statistical distribution of the fatty acids in the triacylglycerides have been developed [3]. As examples of these, the hypotheses that the arrangement of fatty acids in the tryacylglycerides follows a minimum distribution [3], a random distribution [4], a restricted random distribution [5], Gunstone's distribution [6] and a 1,3-random-2-random distribution [7, 8] can be mentioned. Chromatographia Vol. 40, No. 9/10, May 1995 131309-5893/95/05 0557-06 $ 3.00/0 Original 9 1995 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH

* Symbols proposed by authors 557

be achieved. It must be observed, however, that on both non-polar and polarizable columns there is no discrimination between isomeric triacylglycerides such as POO and OPO. Indeed considering only the nature of the fatty acids, while not distinguishing optical and positional isomers, the number of possible triacylglycerides (N), is: N - (n3 + 3 n 2 + 2n) 6 where n is the number of fatty acids present in the sample [13]. Thus, a sample containing 10 fatty acids as part of its composition can yield 220 different triacylglycerides, which makes a vegetable oil a complex sample. In this case, the identification of triacylglycerides becomes a difficult process, in which the number of possible structural forms is very large compared to the number of fatty acids present. Several approaches to identify the triacylglycerol composition of vegetable oils, analyzed by GC, have been proposed [11, 12], but a method for providing a quick estimate has not been previously reported. With the objective of overcoming this problem, a computer program capable of providing the triacylglycerol composition of vegetable oils from fatty acids composition was developed in this work. (1)

Analysis of Triacyiglycerides by HRGC

Solutions of several vegetable oils, diluted in n-heptane (2.0 mg. mL-1), were analyzed by H R G C using a Hewlett-Packard 5890 Series II Gas Chromatograph equipped with a split injector at 360 ~ and a flame ionization detector (FID) at 380 ~ The injection volume was 1 IxL and the split ratio 1 : 30. Hydrogen was employed as carrier gas at 100 KPa. Nitrogen waS used as make-up gas at 20 m L . min- 1. Data was collected with an HP 3396A integrator. Analysis by HRGC on non-polar columns employed a 6 m 0.25 m m x 0.08 pan1 LM-5-HT capillary column (5 % phenyl polysiloxane) donated by L & M Scientific Instruments (S~o Carlos, Brazil). Analysis by H R G C on polarizable colunUas employed a 25 m 0.25 m m 0.1 gm OV-17-OH capillary column (50 % methyl, 50 % phenyl polysiloxane) from RESCOM (Kortrijk, Belgium). For H R G C on the non-polar column, the temperature was maintained isothermally at 350 ~ and for H R G C on the polarizable column, the temperature was programmed at 330 ~ for 1 rain, then increased to 340 ~ at 1 ~ 9min- 1, and maintained at in this temperature for 9 min. These analyses were made without column compensation. Only for peanut oil, analyzed on a polarizable column, the analysis was made with column compensation; the oven temperature programmed at 340 ~ for 1 rain, then increased to 360 ~ at 1 ~ and maintained at in this temperature for 9 minutes.

Experimental Vegetable Oils

Peanut (Arachis hypogaea L.), olive (Olea europaea L.), bacaba (Oenocarpus distichus M.) and patua pulp (Oenocarpus bataua Mart.) oils were studied. They were obtained either by Soxhlet extraction with nhexane (peanut, bacaba and patua pulp oils) or from commercial sources (olive oil).

Computer Program
The computer program is based upon equations which calculate the molar percentage of triacylglycerideS present in a vegetable oil [15-17]. According these equations, if A, B and C are the molar percentages of fatty acids A, B and C, then the molar percentage of triacylglycerides containing only one acid such as the fatty acid A is, A3 % AAA - - 10,000 (2)

Analysis of FAMEs by HRGC

The fatty acid methyl esters (FAMEs) were prepared according to an adaptation to the micro-scale of the method described by Hartman and Lago [14]. Chromatographic analyses were performed on a HewlettPackard 5890 Series II Gas Chromatograph equipped with flame ionization detector (FID) and split/splitless injector. The inlet system was used in the split mode with a split ratio of 1 : 30. Hydrogen was employed as carrier gas with a linear velocity of 40 cm 9s- 1. Nitrogen was used as make-up gas at 20 m L . min-1. The injector and detector temperature were 280 ~ and 300 ~ respectively. A LM-100 capillary column with 25 m x 0.32 mm x 0.3 txrn coated with Carbowax 20M (L & M Scientific Instruments, S~o Carlos, Brazil) was employed at an isothermal oven temperature of 190 ~ The injection volume was 1 IxL Data were collected with an HP 3396A integrator. 558

the molar percentage of triacylglycerides containing two acids such as A and B is, % AAB 3 x A2B
-

O)

10,000 and the molar percentage of triacylglycerides containing three acids (A, B and C) is, %ABC=6XAxBxC 10,000 (4)

In addition, the fact that several investigatiors using the lipase hydrolysis technique have stated that, for vegetable oils, the C-2 hydroxyl group is preferentially acylated by the most highly unsaturated fatty acid, and the C-1,3 hydroxyl groups are subsequently acylated by the remaining acids including any unsaturated acid not Chromatographia Vol. 40, No, 9/10, May 1995 Original

Table!I.

Fatty acids composition of vegetable oils Bacabaoil Patua pulp oil (% Area) (% Area)
0.1 -

FAME Olive oil Peanut oil (% Area) (% Area)

M Pt _ _ _ _

P Hx Po Mg Hp S O V L Ln A Ga
Be
...._.

10.7 0.1 0.6 0.1 0.2 3.0 73.0 2.0 8.7 0.8 0.5 0,3
_

11.7 3.5 40.8 0.5 36.8 1.5 0.7

3.8

0.3 15.8 0,7 29 68,6 1,9 8.8 0.7 0.1

0,1
-

0.3 12.3 0.1 0,5 0,1 0.1 3.2 77.8 1,6 3.6 0.4 -

Lg

0.7

required at the C-2 position of the glycerol moiety [10, 18, 19] was taken into account. We have also considered that the C-3 hydroxyl group contains a fatty acid with a higher degree of unsaturation than those occupying the C-1 hydroxyl group [20]. If the C-1,3 groups are acylated by fatty acids with same degree of unsaturation, then we have assumed that the acid with longer chain length is acylated at the C-3 hydroxyl [21]. From these considerations, and using the specified equations, a program for computation of the triacylglycerol composition of vegetable oils, was made in TURBO-PASCAL language (version 5.0). To establish the validity of the computer prediction of triacylglycerol composition of the vegetable oils, the values obtained by the computer method were compared with those obtained by H R G C analysis by means of the calculus of the correlation coefficient (r).

Results and Discussion

r
~oVlle~netable

ame) /

T
"Calculate the'TAG I Composition [ t'or non-polar HT-CGC] /

ntualexcluded/ i Calculateall possible J TAG groups by I polarizable /

- CGC

ShorthanddesignationI

r non-polar / T-CGC /

ntual exitU~=~]

I r~n-po~ HT-CGCl

-T
F,a~ Acids present/
Combinations

Calculate all possible TAG I

l
I~igure 1

Plow diagram for computer program.

Fatty acids present either free or esterified (as in methyl esters or in acylglycerides) are represented either by the abbreviations of their most common name or by a shorthand designation with the number before the colon specifying the number of carbon atoms and the number after the colon specifying the number of double bonds in the fatty acid. The position and the configuration of the double bonds are also represented. Table I shows some of these representations. The identification of FAMEs and triacylglycerides was made using this convention. The fatty acid composition of the oils, obtained by FAMEs analysis, are shown in Table II. The percentage composition of each acid was calculated by normalization of the peak area. The determination of the triacylglycerol composition of these oils was calculated using the fatty acid composition data (Table II) substituted into the computer program developed above. A flow diagram representing the computer program is given in Figure 1. The program can be used to calculate the triacylglycerel composition of oils to a maximum of 22 different types of fatty acid in their composition. This means that 2024 different triacylglycerol combinations can be obtained using the maximum memory of the program. This fact is not a significant limitation because, generally, less than 15 different acids appear in the vegetable oil compositions. The triacylglycerol composition of these oils, from the computer determination and by H R G C on non-polar and polarizable columns, are shown, respectively, in Tables III and IV. The identification of triacylglycerides by groups with different numbers of carbon atoms (carbon number determinations) and by different combinations of saturated and unsaturated fatty acids (separations on

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T a b l e III. C o m p a r i s i o n o f t r i a c y l g l y c e r o l c o m p o s i t i o n b y n o n - p o I a r H R G C a n d b y c o m p u t e r p r o g r a m developed here. Triacilglycerides (carbon number) Ts0 T51 T52 T53 T54 T56 T58 Tr0 * P e a n u t oil GC* CP* 3.8 . 26.6 52.6 8.1 6.9 2.0 3.4 . . 23.6 56.2 6.7 8.2 1.9 . 28.0 0.5 65.9 1.9 . . 26.3 0.7 67.7 1.9 . . . . O l i v e oil GC CP 5.7 3.4 B a c a b a oil GC CP 6.7 0.7 39.1 . 53.0 0.5 . . 6.8 0.6 34.4 . 57.8 0.4 . . . . . . 62.2 65.5 P a t u a p u l p oil GC CP 4.4 0.9 32.5 4.4 0.8 29.3

P e r c e n t a g e c o m p o s i t i o n o b t a i n e d b y g a s c h r o m a t o g r a p h y ( G C ) a n d by c o m p u t e r p r o g r a m (CP).

T a b l e I V . C o m p a r i s i o n o f t r i a c y l g l y c e r o l c o m p o s i t i o n o b t a i n e d by p o l a r i z a b l e I t R G C a n d by c o m p u t e r program developed here. Triacilglycerides P e a n u t oil GC CP 1.5 . 2.7 . 0.6 7.3 0.9 14.2 5.6 0.4 1.9 10.5 3.3 18.3 16.5 . 2.6 0.8 0.7 1.7 1.2 0.7 0.8 0.6 0.3 1.2 0.6 2.1 1.6 0.4 0.6 0.4 1.7 . 1.5 . . 1.1 7.0 a 10.9 4.8 0.6 2.6 10.6 b 20.6 17.1 . 5.1 1.3 2.0 e 1.8 . 1.3 d 0.3 0.6 . 2.5 e 3.6 1.6 0.6 0.7 0.3 . . 1.1 23.0 . 6.0 0.8 0.3 0.6 0.6 5.5 42.8 . 12.0 2.5 . . . 0.5 . 0.4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 18.5 . 5.5 0.9 0.2 0.4 0.5 5.3 43.9 . 15.0 3.1 . . 0.4 . 0.9 . 02 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O l i v e oil GC CP 3.0 . 0.9 2.6 . 0.6 . 1.7 0.5 1.2 31.1 . 6.8 0.9 . . . . 4.4 35.6 . 9.8 1.8 . . . . . . . . . . . . . . . 1.3 0.5 2.0 24.5 . 7.1 1.1 . . . . 4.6 37.0 . 13.6 2.8 . . . . . . . . . . . . . . . . . . . . . . 4.1 0.4 7.0 1.1 B a c a b a oil GC CP 6.2 5.5 P a t u a p u l p oil GC CP 4.1 0.4 0.8 1.4 30.2 3.6 . . . . 5.3 49.7 6.2 51.7 3.7 0.5 0.6 1.9 23.9 3.3 -

POP PPoP PLP PtOO POS POO PLS PLO PLL PLnO MgOO HpOO POA SOO OOO SLO OLO OLL OLnO LLL OLnL POBe AOO PLBe OOGa ALO OLGa ALL GaLL SOBe BeOO SLBe BeLO BeLL LgOO LgLO LgLL a b e d e Sum Sum Sum Sum Sum of of of of of

. . . .

POO and PLS percentage composition. OOO and SLO percentage composkion. AOO and PLBe percentage composition. OLGa and ALL percentage composition. BeOO and SLBe percentage composition.

560

Chromatographia

V o l . 40, N o . 97t0, M a y 1995

Original

IOLO

kU~t ,t.at~

5 1'0 t Imln) l~igut, 2 e Chronaatogramof peanut oil on non-polar LM-5-HT column.

0 Figure4

10

1'5

20 t (rain)

Chromatogram of peanut oil on polarizable OV-]7-OH column.

rra

T~

s
~gere 3

;o

;6

t,

Chromat0gram of bacaba oil on polarizable OV-17-OH column.

polarizable stationary phases), were made by comparing the percentage data from HRGC with those provided by the computer. The data in Table III and IV show that the proportions of each component calculated by computer, are close to those obtained by chromatographic analysis. Thus, identification of the triacylglycerides present in these vegetable oils was made. As an example, the chromatograms obtained for the analysis of Peanut oil on the non-polar LM-5-HT column (Figure 2) and the analysis of Bacaba oil (Figure 3) and Peanut oil (Figure 4) on the polarizable OV-17-OH column are shown. The final data provided by the computer program, simulating the separation of triaeylglycerides on polarizqble columns, do not provide the separation of some triacylglycerides such as POO and PLS, which are present in peanut oil. Thus, the percentage composition of POO, provided by computer, represents the sum of the percentual composition of POO and PLS. However, as mentioned earlier, on a polarizable column, triacylglycerides with the same carbon n u m b e r and degree of unsaturation, such as POO and PLS, can be separated owing to polarity differences in the triacylglycerides. Thus, the computer determination of these triacylglycerides in the oil should be made with the help of Table II (see Figure 1), provided by the computer

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Table V, Correlation coefficients (r) for triacylglycerol composition obtained for vegetable oils by computer and by the HRGC on non-polar (LM-5-HT) and polarizable (OV-17-OH) capillary columns. Capillary column LM-5-HT OV-17-OH Peanut oil Olive oil Bacaba oil Patuapulp oil (r) (r) (r) (r) 0.9954* 0.9993** 0.9711"* 0,9918"* 0.9916'* 0.9802** 0.9968* 0.9890**

References
[t]
E. Geeraert, P. Sandra, L High Resolut. Chromatog. 8,

415 (1985). [2] N. R. Antoniosi Filho, t 7. M. Laneas, J. Am. Oil Cheril. Soc. 70, 1051 (1993). [3] A. P. Doerschuck, B. F. Daubert, J, Am. Oil Chem. Soc. 25,425 (1948). [4] E.W. Eckey, in "Vegetable Fats and Oils", Reinhold Publishing Corporation, New York, 1954, p. 43. [5] A . R . S . Kartha, J. Am. Oil Chem. Soc. 30, 326 (1953). [6] t7. D. Gunstone, Chem. Ind., 1214 (1962). [7] R.J. Wander Wal, J. Am. Oil Chem. Soc. 37, 18 (1960). [8] M. II. Coleman, J. Am. Oil Chem. Soc. 38, 685 (1961). 1, [9] G . K . Chacko, E. G. Perkins, J. Am. Oil Chem. Soc. 4 843 (1964). [10] E.G. Perkins, J. Am. Oil Chem. Soc. 42, 1032 (1965). [11] E. GeeraerL D. De Schepper, J. High Resolut. ChronaatO" gr. 5, 80 (1"982). [12] E. Geeraer, D. De Schepper, J. High Resolut. Chromatogr' 6, 123 (1983). [13] M. Farines, R. Soulier, J. Soulier, J. Chem. Educ. 65, 464 (1988). [14] L. ltartman, R. C. A. Lago, Lab. Pratt. 22, 475 (1973). [15] R.J. Vander Wal, J. Am. Oil Chem. "Soc.40, 242 (1963). [16] C. Merrit, C. MerritJr., M. Vajdi, S. G. Kayser, J. if' Hallyday, M. L. Bazinet, J. Am. Oil Chem. Soc. 59, 42;[ (1982). [17] D. Swern, in "Bailey's Industrial Oil and Fat ProduCtS", John Wiley & Sons, New York, vol. 2, 1982, p. 150. [18] C. B. Sharma, G. C. Martinez, J. Am. Oil Chem. Sor 49, 229 (1972). [19] F. D. Gunslone, R. J. ltamilton, t7. B. Padley, M. L Ourr J. Am. Oil Chem. Soc. 42, 42 (1965). [20] G. Sempor~, J. B~zard, J. Am. Oil Chem. Soc. 68, 70;[ (19'21). [21] R. Maurin, K. Fellat-Zarrouck, M. Ksir, J. Am. Oil chert. Soc. 69, 141 (1992). [22] J.A. Singleton, IL E. Pattee, J. Am. Oil Chem. Soc. 64, 534 (1992). [23] J.C. ltokes, R. E. Worthington, J. Am. Oil Chem. Soc. 56, 953 (1979). [24] T. G. Toschi, W. W. Christie, L. S. Conte, J. High Resolut' Chromatogr. 16, 725 (1993). [25] N. R. Antoniosi Filho, F. M. Lan~as, unpublished results (1994). Received: Sep 6, 1994 Revised manuscript received: Jan 2, 1995 Accepted: Feb 1, 1995

* Significant between 99 % and 99.9 % ** Significant up to 99.9 %

program. In this table all possible triacylglyceride combinations in the oil are found. T h e identification of some triacylglycerides ( P O d , PLS; O O O , SLO; A d O , PLBe; O L G a , A L L ; B e O O , S L B e ) in peanut oil was made using this artifice. It will be observed that for the analysis of peanut and olive oils on a polarizable column, the identification of the triacylglycerides obtained was the same as that reported by other authors [1, 22-24]. The values obtained by computer were compared with those obtained by H R G C by means of the calculus of the correlation coefficient (r). The data obtained arc shown in Table V. The results obtained demonstrate the high correlation between the H R G C data and the computer predictions. In our laboratory, the utilization of this computer program permitted identification of all triacylglycerides present in more than 30 vegetable oils and vegetable oil blends with high levels of unsaturated fatty acids, analyzed by means of H R G C on non-polar and polarizable columns [25]. Unfortunately, the computer programs p r e s e n t e d some difficulties in the identification of the triacylglycerol composition of fats.

Additional research is being carried out to explain the

process of distribution of the fatty acids in such samples.

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