Gas Chromatographic Determination ofTatty Acid Compositions Horacio Heinzen a n d Patrick Moynal Catedra d e Farmacognosia, Facultad d e Quimica, Avda. General Flores 2124, Montevideo. Uruguay Antonia G r o m p o n e Cia. Bao S.A., Real 4338. Montevideo. Uruguay
Gas chromatoeranhic ., . determinations are well established a s part of undergraduate l a h ~ m ~ o experiments ry un instrumental analvsis 1161. One ihiwtion 151 leveled at some of the suggested ekperimeits is t h a t the determinations d o not include the derivatization s t e ~tsh a t are almost universal in anv "real life" situation, thus giving the students a n erroneous feeling of sim~licitv. . . ~ e b r o p o s ae n experiment t h a t does have a derivatization step using readily available reagents; t h a t requires limited manipulative skills, centering the attention on the method itself; t h a t can he completed within the time constraints of a normal laboratory course; and t h a t "investigates" materials t h a t are easy t o acquire, and are of great technical and biological potential (6, 7). A t the same time, t h e technique is suited as an introduction to programmed temperature use, one of t h e true potentials of gas chromatography (8).
Table 1. Composition a of Coconut 011 Sample
C m
C12:0
CWO
CWO
Cleo
C18:l
standard coconutb
18.1
32.2 41.2
7.0
16.8 5.9
2.2
27.0
5.6
C ~ O
33.1
GLC on OV-I. Far operating MnditlOnJ see text. Caconui oil was from Paraguay. It also contained Cwa.7.6%. and Cia:*, 3.6%( 14. wlw by
3
Table 2.
Composklon a of Edible Oils from Uruguay
standard A
18.3 31.8 16.4 . . . . . . 7.3
cw cw C18:~C20:0 C18:3 6 2 2 ~ . . . . . . . . . 32.8 . . . . . . 3.7 19.5 67.0 ... 1.7 0.5
F G
...... ......
3.1 3.2
Sample C I ~ Oc12:0 CIW
Experimental Instrumentation The gas chromatographs used were a Pye Unicam 104 and a Shimadzu GC-GAMPr, both with Dual FID detectors. Columns used were OV-15%on Diatomite CAW DMCS 100-120mesh, glass, 1.5 m length, 3 mm i.d.; and SP-2330 10%on Chramosorb W AW 100-120 mesh, stainless steel, 1.80-m length, 3-mm i.d. Syringes were either Hamilton 701N or SGE 10 wl. Thin-layer chromatography plates were Silica Gel G (Merck), 20 X 20 cm, 0.25-mm thickness, prepared by the usual method. Acid standards were Fluka purum, including capric (Cleo), lauric (C12:o). palmitic (Cle:~),and arachidic (C2o.o). Reagents used were: diethyl ether (BDH), Analar); KOH pellets (May-Baker, analytical grade); methanol (Merck, analytical grade);methylene chloride (BDH, Analar); HC1 (Merck, analytical grade); HzSOn (Merck, analytical grade); petrol-ether (ANCAP 60-80);nitrogen (AGA, pure); N-nitrosomethyl urea was prepared previously following Vogel(9), and kept in the freezer until used. Materials used were coconut oil from Paraguay (101, and the following vegetable oils available in Montevideo: Gran Visir-SAIM;Optimo, El Torero, Sibarita, Uvasol, Primor-COUSA; Manzanares-Manzanares. Derivatization of Free Acids ( 11) Duration of experiment, 15 min. To he carried out in a fume eupboard. See notes in ref. (12). Drop one KOH pellet into a test tube containing 2 ml dry diethyl ether. Add 30-40 mg (one spatula tip) of N-nitrosomethyl urea into the tuhe and mix vigorously (a Vortex mixer is especially useful),until an intense yellow color develops. Let stand for a few seconds, and pour this solution into a second test tuhe with 10 mg of a mixture of free The fatty acids in the following ratio: Cleo, 1; C m , 2; C16:o. 1;C~o.0~2. vellow color of the solution must ~ e r s i sat t the end of the reaction. should it fade, add a second diazbmethane preparation to the free fatty acid test tube. Derivatization of Oils ( 7) Duration of experiment, 20 min. Dissolve 1drop of oil in 0.5 ml diethyl ether. Add 1 ml of 0.5 N MeOH-KOH solution to this oil solution, and shake. After 10 min at room temperature, add 1.0 ml of 0.1 N HC1 and shake. The resulting solution can be gas-chromato-
6.1 10.6
22.3 67.4 19.1 59.9
0.1 6.5
0.2
...
0.7 0.3
' r i a by U C on SP-2330 Fw operating condltlons see text Relative mention times: Clmo,capric:0.25: Cq2:~.Isuric: 0.37: C,ro. palmitic: 1.00: Cleo. stearic: 1.69: C ,.,. oleic: 1.98; C1.:2. linolsic: 2.50: Czoo. arachidic: 2.87: CT.~,
linolenic: 3.25:Csea. behenic: 4.70.
graphed directly, or, better, the methyl esters can be extracted by washing the neutralized solution with petrol ether (3 times with 1.0 ml each), concentrating the combined ether extracts under nitrogen to 1 ml and then injecting. Esterification Control by TLC Duration of experiment, 30 min. The derivatized fatty acids and the methyl esters derived from the oils can be checked against standards of the underivatized oils and one of the free fatty acids. The samples are spotted on a Silica Gel G TLC plate, and developed with CHzC12-MeOH (99:l). The developed plate is sprayed with HzS04 sq. 50%,and charred for visualization. Underivatized fatty acids have an Rr of approx. 0.18, triglycerides 0.73, and methyl ethers 0.85. Gas Chromatography ( 11, 13) Duration of experiment, 30 min. Operating conditions for OV-13%: carrier gas (Nz)flow, 30 mllmin; injector and detector temperature: 300°C; initial oven temperature, llO'C; final oven temperature, 250°C; initial time 0 mi"; program setting SoCImin. Operating conditions for SP-2330 10%:carrier gas (N2) flow, 20 mllmin; injector and detector temperature: 250°C; column oven temperature: 185% Results and Discussion T h e results obtained with the coconut oil sample are shown i n Table 1a n d the results obtained with t h e vegetable oils in Table 2. The use of N-nitrosomethyl urea in the conditions proposed obviates t h e distillation of the diazomethane t o be used, Author to whom correspondence shouid be addressed. Volume 62
Number 5
May 1985
449
eliminating most of the risks involved in working with this reazent (9.12). We therefore avoid the use of other alternative routes for diazomethane production (11).Due to the use of less than 10 pg of N-nitrosomethyl urea per standard preparation, it is highly improbable that risk situations will arise in afairly organized lab. In our course we have managed to work with groups of up to 27 students simultaneously without problems. From the data in Table 2, it is possible to determine the raw materials used in the production of the edible oils under study (6). For example, sample A is pure sunflower oil, whereas sample G is pure soybean oil. On the other hand, the other oils are mixtures such as sunflower and peanut (sample B), soybean and peanut ( C ) ,sunflow and olive (Dl, or soybean and grapeseed (E). T h e experiment can be applied to other triglycerides (e.g., animal fats) or other sources of free fatty acids (SO~DS).
T ~ OV-l P rxperimrnr is better adapted to programming and samples rich in saturated h r t s acids. whereas the 5-2:1:10 experiment is more suitable for samples, such as vegetable oils, that are rich in unsaturated fatty acids. The experiment is simple to follow and accomplishes two objectives: 1) It demonstrates two different derivatization techniques, also exemplifying differing reactivities for the suhtrates to he ana-
lyzed
450
Journal of Chemical Education
".
2) It shows the imorovement of results usine oroerammed tem" oerature control over isothermal ooeration in the OV-l eaoer-
temperature experiment it is possible to identify specific acids solely by their retention time, without recourse to direct comparison with a complete set of standards. For coconut oil this is done for myristic and stearic acids. With the operating conditions eiven for the ex~erimentthe time delav from one rnernbe;'of the homolugbus SPTIPS to the nrrr is'prartirallv cunstlnt. The 1:2:1:2 ratioolstandardssirnplifirstheir idrntification. Literature Cited 111 . . Ewine.G. W.."lnatrumental Methodsof ChuniealAoalvaia."3rd.od..Mffirsw-HiU. . . Y~.~,IW. (2) Dabrio, M.V., "CromatoprdmdeCases," Alharnbra, Madrid, 1911. (3) Pattison,J. B.. "A Pmgrammcdlntmdtmdcticticti to Gas-Liquid Chmmatography." H w d d 8 Sons.London, 1969. (4) Benaon, G. A,, J. CHeM. EDuC,59,344 (1982). (5) Rudzimki, W. E., and Beu, S., J. CHBM.EDUC.,59.614 (1982). 161 . . Hilditch. T. P..and Williams. P. N.."The Chemical Constitution of Natural Fats." chap& & b.don, im. (7) Litchfield, C., "Analysi8ofTriglyeerjdes."AeaddmieP~eee.New York, 1972. Sci. 13,505 (1975). (8) 1verson.J. L., and Sheppard,A. J,J.Chr~motagraph. (9) Vage1.A. I., "ATertbwkof PmctidOrganicChemistry," 3rd. d.,Iangman,Iandan.
NG
