Analysis of the Triglycerides of Some Vegetable Oils Marie Farines, Renee Soulier, and Jacques Soulier Laboratoire de Chimie Organique des Substances Naturelles, Universite, Avenue de Villeneuve. F66025 Perpignan, France Lipids, whether of animal or vegetableorigin, consist 85 to 95%uf triglycerides, edters of fatty acids and glycerol:
The qualitative and quantitive analysis of constituent fatty acids is important in the characterization of an oil or fat; in effect, numerous properties of the oil are linked to the nature of fatty acids: viscosity, melting point, dietetic properties, stability when heated (use for frying), oxidability (aptitude to rancidity), siccativity (use as solvent for paints), etc. However, it is sometimes observed that oils or fats, although having similar fatty acid composition, have fairly different properties; this can be attributed to the fact that fatty compounds are essentially composed not of free fatty acids but of triglycerides. Their properties depend not only on the nature of the fatty acids but also on the way in which these fattv acids are esterified with elvcerol -. to form the triglycerides. Indeed. if one considers the total number of trielycerides thar could be built up with n different fatty acid;, it is N = n'. including trirlvcerides differing t'rom each other hv the nature of tKe fatty acids, the po&ional isomers, and the enantiomers: no simple method currently allows such an analysis to be performed. If one does not iake into account the optical isomers, the number of possible triglycerides is then h" = InZ n'112. I t one considers onlv the nature of the fatty acids; the number of compounds is^' = (n3 + 3n2 2n)/6, and a recent analytical technique allows their separation, identification, and quantification by a convenient HPLC method (1-8). For instance, let us consider a triglyceridic mixture formed by the glycerol esters of three different fatty acids A, B, and C; one can theoretically separate by HPLC 10 different fractions: AAA, BBB, CCC-one isomer in each case; AAB, AAC, ABB, ACC, BBC, BCC-three isomers in each case (that is, a couple of enantiomers and a positional isomer); ABC-six isomers (that is, three positional isomers and their enantiomers).
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Teaching Objective The aim of this article is to show students that triglycerides, which are the main constituents of natural lipids, consist of a mixture of different compounds, depending on the total number of fatty acid constituents. The students will perform a GC analysis of fatty acids methyl esters, then a HPLC analysis of triglycerides (TG). They will then search for all the possible structures of TG and compare the results with the actual composition of the oil or fat. Principles of Manipulation Separation of triglycerides from the oil. The triglycerides can be separated rapidly from the other constituents of the oil by chromatography on a silica column, the eluant being benzene. This separation is not absolutely necessary, but the
464
Journal of Chemical Education
HPLC chromatograms thus obtained are not perturbed by secondary peaks. Analysis of fatty acid methyl esters by GC. The fatty acids methyl esters (FAME) are prepared by transesterification of an aliquot of the triglyceride fraction by a solution of sodium methylate in methanol, acidification and extraction of the FAME by methyl tertbutyl ether (9); the organic solution can then be used directly for the GC analysis. Identification of the FAME can be achieved by comparison with standards chromatographed by the instructor a t the beginning of the session. Analysis of triglycerides by HPLC and interpretation of chromatograms. A fraction of the triglyceride solution is fractionated by isocratic HPLC using propionitrile as solvent. The chromatography is complete in about 20 minutes and reveals a number of peaks (around lo), which are characterized by their retention times. For identification of the different triglycerides, each fatty acid is assigned a one- or two-letter abbreviation, P for palmitic acid, S for stearic acid, 0 for oleic acid, L for linoleic acid, and Ln for linolenic acid. Thus OLL represents oleodilinolein. and so forth. The instructor determines in advance the retehtion times (R7')of some homogeneous triglycerides: 000. PPP. SSS, LLL. LnLnLn. The theoretical retention rime ot a given rrrgls~erideis easllv calculated by adding the contrihutlons of the constnuent ratty acids. If one considers a homogeneous triglyceride such as triolein, let us define log RT(0) = % log R T ( 0 0 0 ) . The logarithm of the retention time of any triglyceride is the sum of the logarithms of the retention times of each of the three constituent fatty acids (2,101. For example, log RT(P0L) = log RT(P) +log RT(0) + log RT(L) log RT(SS0) = 2 log RT(S) + logRT(0) Knowing the fatty acid composition of their oils, the students make a list of all the possible triglycerides ((n3 3n2 2n)/6), that is, 20 for four fatty acids, 10 for three fat,@acids, etc. The theoretical retention time for each triglyceride is then calculated, and the triglycerides are arranged by increasing retention times. The students can also calculate the partition numbers (PN = number of carbon atoms in the fatty acids minus twice the number of double bonds) and observe that the triglycerides are eluted in approximately the order of their partition numbers (10,lI). The students then establish a correlation between the theoretical retentiun times and thuse trf the peaks they haw actually obtained. The precision is ahour +0.2 min. It s h ~ u l d be noted that not all of the possible triglycerides are actually observed.
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Results Four vegetable oils have been studied by our students in "Applied Chemistry and Biology" at Perpignan University; grapeseed, cornseed, rapeseed, and sunflowerseed oils. The fatty acid composition of each oil is given in Table 1. These oils were chosen because they contain three or four main fatty acids found frequently in natural products: palmitic acid (abbreviated C E O , i.e., 16 carbon atoms and no double bond), stearic acid (C18:0), oleic acid (C18:1, i.e., 18 carbon atoms and one double bond), linoleic acid (C18:2), linolenic
Table 1.
Fanv Add Comoosition of Some Seed Oils
Palmitic acid Staaric acid Oleic acid Linoleicacid Linolenic acid
RT
grape
(mi")'
(%)
5.0 8.8 9.5 11.1 13.1
5.8 3.2 15.0 75.9 Trace
corn (%)
rape
sunflower
(%)
(%I
10.2 Trace 30.3 59.5
4.3 Trace 63.3 23.8 8.5
6.1 3.8 22.5 67.5 Trace
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FAME retention timer refer to CondHions given in
experimental psrt.
acid (C18:3). Figure 1shows a typical HPLC chromatogram obtained by o u r s t u d e n t s for t h e triglycerides of two oils. T h e retention times of the homogeneous triglycerides L n L n L n , L L L , 000, P P P , SSS were measured directly on commercial samoles, and t h e theoretical retention times of t h e other triglycerides were calculated as indicated above. T h e results a r e summarized in T a b l e 2. A total experimental t i m e of about three hours is needed for each grouo of two students to oerform the experiments. F o r the 6est &e of equipment, grbups were s t a r t e d at onehour intervals so that t h e y could u s e each of the instruments, HPLC and GLC, for about o n e hour. Experimental
Rapeseed
Corn
Soiutions
.
Soluents. Prooionitrile. benzene.. ~ e t r o l e u mether. methvl t-butvl ether, methanol'.klli~fth~cesulventsnreanhydrous.'~nrpi~~n1tr11e~~ filterrd and dcyassrd by ultrasonic treatment [under r e d ~ ~ t prescl sure. Tronsesterification agent. Solution of sodium methylate approximately 0.2 N in methanol prepared by dissolving 0.46 g ofsodium in 100 mL absolute methanol; sulfuric acid 1 N in aqueous solution. Standards. Triglycerides 000,PPP, SSS, LLL, LnLnLn; methyl esters of oleic. oalmitie. stearie.' linoleic. linolenie acids (Siema . Chemical Co., Louis, MO). Analyzed oils. Commercial samples bought in a supermarket.
SL
instrumentation IIP1.C. We used a Merrk apparatus equipped \\ith a Waters differential refrsct~,mrtersnd a Mcrck integrating rrcorder: hlerrk Lichrosort,column R P l a 5 om, 25cm long by 1 mm internaldlamcter: 20 *I syringe: 1 "11. min tlowrate. GT. Delsi chrumatograph equipped with a capillary column in?wranacrd with Cnrhuwnx 21lhl. 25 m lmr and 0.G n m inrcrnal diameter and with an injector with evapora&r. Analysis conditions: 190°C oven, 250°C injector, 250 *C detector, helium gas vector at a pressure of 0.7 bar; 1 r L syringe; Hewlett Packard integrating recorder.
Method HPLC chromatograms of the triglycerides of two seed oils. Letters refer to the triglycerides mentioned in Table 2.
Table 2.
LLnln LLLn LLL OLL~ OLL ooLn PLL
0) (k) (a)
(I) (b)
(m) (C)
LOO - -
(dl ~-,
POL SLL 000 PPL PO0 SOL PPO PSL PPP SO0 Others
(e) (e)
(9) (1) (h) (i)
Triglycerldic Composition 01 Some Seed Olb'
@in)
Exp. RTd (min)
% TOe
PN
grape
% TG corn
% TG rape
38 40 42 42 44 44 44 46 46 46 48 46 48 48 48 48 46 50
4.14 4.71 5.37 5.77 6.57 7.07 7.34 8.05 9.00 9.12 9.87 10.05 11.03 11.30 12.32 12.62 13.76 13.85
4.21 4.84 5.36 5.61 6.56 8.90 7.55 7.93 8.91 8.91 9.95 10.4 11.32 11.50 12.36 12.87 13.77 13.60
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-
0.3 2.5
39.3
21.4
Th. RTC
Triglyceride
Purification and analysis of the triglyeerides. Precision weigh 0.1 g of oil. Into a 30-cm-long hy 12-mm-diameter chromatographic
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26.6
27.6
sunfl.
-
-
26.9 28.5
-
-
6.4 10.5 10.5
11.0 8.7
11.7 15.9
27.6
14.4
10.9
4.5
-
3.4 4.8 2.3
29.9
-
-
7.6 11.0
2.9
-
-
0.9
1.9
-
-
-
-
0.6
-
0.5
1.0
(x)
% TG
0.3 0.9
~etmtiontimes of standards:~ n ~ n 3.64 ~ n .min: LLL. 5.37 min: o w . 9.67 min: PW. 13.76min; SSS, 27.27 min PN = panition number. iTh. RT= theorical retention time. b E ~ pR . T = experimental retention time. ' TG = friglyceride. Volume 65
Number
5
May 1988
465
column, transfer 5 g of silica (Merck, Kieselgel40) in suspension in 20 mL of petroleum ether. Elute the supernatant liquid, and add the oil dissolved in 1mL of chloroform on the silica and elute with 30 mL of benzene. Evaporate the solvent under reduced pressure, and dissolve the triglycerides in 0.2 mL of chloroform. An aliquot of 20 pL is sufficient for HPLC analysis. Esterificationof fatty acids. After studying theTG by HPLC, the total fatty acid composition of the TG can be determined by GC analysis. The chloroform is evaporated and the residue is dissolved in 2 mL of methyl tert-butyl ether. Sodium methylate (1 mL) is added; the mixture is mixed well for 1 min, allowed to settle for 2 min, and then neutralized with 0.2 mL of 1N sulfuric acid. After mixing, 3 mL of water is added, and the mixture is mixed and again
allowed t o settle. The FAME contained in the organic phase analyzed by GC; inject 0.5 pL in the chromatograph.
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6. singleton, J. A ; pat&, H.W.J. Am. Oil c h h . Soc. 1984.61.161. 6. Phillios.F.C.:Erdshl. W.L.;Priveft.O.S.Lioids1984.19.880.
An Automatic Controller for a Water Still The inatallation ofan audible high-unrer alarm for the receiver ot our steam-hcnlrd water .;till has eliminated nearly a11 of the once-common flocdings due LO uverflo\r 1. The imlg cxreptims have been when absence of personnel hns left the alarm xranal unheard. The control 9ytpm has now been redesigned to hark up I he nudlhlc warning with autonintic shutc~ff of steam. Normally closed bypass valve BP can be opened if reversion to manual control is desired. Otherwise steam can reach the still only when solenoid valve SV is energized2. This occurs when START button S2 is pressed. Steam then flows until the distilled water level in the receiver has risen to approximately 1mm above the bottom edges of the electrode pair E. Cutoff can also be achieved by pressing STOPITEST button S1.
Automatic Controller. B, pulsing buzzer; D, 6.24 Zener diode: E, electrodes: F, fuse. 0.25 A: L1.6-V sensitive SPST relay: La, 1 2 4 DPDT relay; P, amber pilot lamp (POWER): p', red pilot lamp (STEAM):ps, 1 2 4 dc power supply: a, transistor MPS-A13: R,, resistor 470 K% R2,variable resistor 1 M n : Ra,resistar(see text); S,, pushbunon switch (STOPITEST):S2.pushbunon switch (START):S3. line switch. Steam system: BP, bypass valve; MV, main valve: SV, solenoid valve. The circuitry, including the 12-V dc power supply PS, is built into a Sir- X 3- X 57h-in. aluminum minibox. The momentary-contact pushbuttons, line switch, fuseholder, and pilot lamps are mounted on the front of the box. All other components, including SENSITIVITY control R2, are mounted internally. Resistor Ra allows the use of a n available 6-V sensitive relay and is not needed for a 12-V version. Momentary depression of the START button causes relay L2 t o pull in and to latch, because movement of the lower pole allows it t o supply current to the relay coil. When the electrodes are bridged by the rising level of water, Darlington transistor Q turns on, causing relay LI to pull in. This activates buzzer B and cuts off the current t o Lz, causing it to drop out. The steam supply is arrested and cannot be restarted until water has been drawn and the electrodes are no longer bridged. Thesystem, designed tocontrol steam, can obviously heapplied toother formsofheating. In essence, the critical level of any liquid that has a t least minimal electrolytic conductance can he sensed and caused to operate almost any final control element.
' Stuck. J. I'.J. C h r m Educ. 1988,62, h14. ' AscoCatalog 822 2A66, ,\utomatir Snitch Co.. $0 Hanover Hd., Flurham. SJ 079.32 John 1.Stock University of Connecticut Stows. CT 06268
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Journal of Chemical Education
