The fatty acid composition of edible oils and fats: A beginning glc

I of Edible Oils and Fats. The Flintkote Company. 10s Angeles, California. I A beginning glc experiment. Edible oils and fats are products of either v...
7 downloads 0 Views 2MB Size
Donald R. Paulson' m o J o h n R. Saranto California Stote Un~versity Los Angeles, 90032 William A. Forman2 The Flintkote Company 10s Angeles, California

1 I

I

The Fatty Acid Composition of Edible Oils and Fats A beginning glc experiment

Edible oils and fats are products of either vegetable or animal origin consisting of triglycerides of mixed fatty acids; oils are distinguished from fats by being liquid a t room temperature. Commercial materials may originate from a single source, e.g., safflower seed oil or bovine depot fat (lard), or may be a blend of materials from different orieins. e.a.. cooking oil. In recent years it has become fas6on&le to advertise the benefits of polyunsaturation in edible oils and fats a s having superior nutritional properties. Consumer Reports ( I ) has recently reviewed the properties and claims of a variety of cooking fats and oils and the nutritional aspects of polyunsaturation. The analysis for the fatty acid content of fats and oils is routinely performed in industrial laboratories by transesterifvinz trirlvcerides t o fattv acid methvl esters and separating t h l -ester mixtures- by gas chr&natography. Fmm its verv i n c e ~ t i o n .gas chromatography (2) has revolutionized lipid analysi'spemitting &alys& t o be made in a few minutes a s opposed t o weeks. Hardly a month passes t h a t there does not appear in the literature a report based on glc analysis of fatty materials. This technique finds application in agriculture, in biochemical and industrial laboratories and yet, in spite of its widespread use, n o organic chemistry laboratory textbook has, to our knowledge, included this technique for fatty acid analysis. However, two biochemistry lab manuals have included separation of fatty acid esters by gas chromatography (3). Excellent reviews of the field are available and some of the more recent ones are quoted below (3, 5). Books by Gunstone (6) and by Chapman (7) give a good introduction t o fatty acid chemistry; the book by Litchfield (8) deals specifically with the analysis of triglycerides. Specifications for the fatty acid content of several oils and fats have been published (9). In a continuation of our efforts t o develop experiments which give the student a feeling for work which is routinely performed in industrial laboratories (lo), we have developed this experiment in which oils or fats are saponified to the acids and esterified by methanol/BFa catalysis. The methyl esters are then identified by determining their Equivalent Chain Length (ECL) and quantitated by peak integration. Experimental Transesterification From the several rapid esterification methods available in the literature we have found the following method to he the simplest and most reliable (I 1, 12). To approximately 100-200 mg of the oil or fat contained in a small test tube is added 4 ml of 0.5 N methanolic sodium hydroxide. The mixture is heated an a steam bath until the fat globules go into solution (3-5 min). To the test tube is added 5 ml of BFa/methanol and the mixture boiled for 2 mi". The BFa/methano1 solution is conveniently prepared by huhhling BF3 into cold anhydrous methanol until a 12.5% wt/v solution is obtained. The mixture has a shelf life of -4 mo if stored in a brown battle. Alternatively, BF3/methanol solutions, in sealed ampoules, are available from a variety of source^.^ 406

/ Journal of Chemical Education

The boiled mixture is transferred into a 125-ml separatary funnel with 30 ml of 30-60°C petroleum ether; 20 ml of saturated sodium chloride solution is added, the funnel shaken vigarously, and the lower layer allowed to separate. The aqueous methanol layer is drained off and discarded. The petroleum ether layer is filtered into a 50-ml heaker and the solvent is evaporated on a steam hath. To this residue is added 0.1 ml of a 50% (wt/v) solution of methyl heptadecanoate in chloroform. The methyl heptadecanoate can easily be prepared from heptadecanoic acid and BFs/methanol solution. The final mixture is now ready for glc analysis with the methyl heptadeeanoate serving as an internal standard. Gas Chromatography We employed a Varian Aerograph Model 920 gas ehromatograph equipped with a thermal conductivity detector and a column consisting of a 10-ft x Ycin. o.d. aluminum tubing packed with 60180 mesh Chromosorh W coated with 15%DEGS (bisethylenegly~olsuccinatepolyester). The experimental conditions were: helium flow-120 ml/min; temperatures: injector-228"C, detector-200°C. column-190°C: detector filament current-150 mA. The sampGs injected were i'n the 2-3-,~1range. A very goad review of syringe handling techniques is available3 from Applied Science Lab., Inc. (13). Other chromatographs are equally well suited and hath TC and FID detectors may be used. Temperature programming, although an asset in precision analysis, leads to excessive confusion in a large laboratory class; hence, we adopted isothermal conditions. Many modifications of the DEGS stationary phase are available from the previously mentioned s ~ u r c e s . ~ Fatty Acid identification and Ouantitation Identification: A mixture of known campasition of high purity fatty acid methyl esters, obtainable from sources q ~ o t e d and ,~ methyl heptadecanoate (17:0) is chromatographed to determine retention times. The 179 methyl ester is not naturally occurring in fatty materials and serves as an internal standard. The mixture should include the fallowing fatty acid methyl esters: myristic (14:0), palmitic (16:0), stearic (18:0), araehidic (20:O). and behemic (22:0) for the saturated series. The following should be included for the unsaturated series: palmitaleie (16:1), oleie (18:1), linoleic (182). linolenic (18:3), and eieosenic (20:l). In order to minimize gas flow differences from day to day and column aging effects, retention values relative to 17:0 were calculated by dividing the distance between the injection point and the fatty acid ester peak by the corresponding value for 17:0. The Equivalent Chain Length (ECL) values for the unsaturated methyl esters were determined in the following manner. A plot of the relative retention values of the saturated methyl esters versus their carbon numbers was prepared on semi-log graph paper. The relative retention values of the unsaturated acids were then determined. The place where these values intersect the previously drawn carbon number line for the saturated esters is taken to he Author to whom correspondence should be addressed. 2Present address: PAVCO, S.A., Bogota, D.E., Columbia, South America. 3Analabs, Inc., 80 Republic Drive, North Haven, Connecticut 06473. Applied Science Laboratories, h e . , P.O. Box 440, State College. Pennsylvania 16801. Supelco Inc., Supeleo Park, Bellefante, Pennsylvania 16823. The Hormel Institute, 801 16th Ave., N.E.. Austin, Minnesota 55912.

Figure 2. Actual chromatogram of fatty acid esters from commercial salad ail

Table 3. Fatty Acid Content of Selected Fats and Oils (wt %) ;5

16

(7

Ew~,.",

.mm

(8 i.n*n

19

Product

20

numa.

myristic myristoleic palmitic palmitoleic stearic oleic linoleic arachidic linolenic eicosenoic hehenic

14:O 14: l(9c) 16:0 16: 1(9c) 18:O 18:1(9c) 18:2(9c,12c) 20:0 18:3(9c,12c,15c) 20: 1( l l c ) 22:O

Experimental Literature values (17) values

14.15 14.75 16.00 16.45 18.00 18.40 19.21 20.00 20.20 20.42 22.00

14.00 14.71 1 6 00 16.55 18.00 18.43 19.22 20.00 20.12 20.36 22.00

T h e n u m h e r before t h e colon indicates t h e n u m b e r of carbon a t o m s i n t h e p a r e n t straight chain f a t t y acid while t h e n u m b e r after t h e colon indicates t h e number of double bonds i n t h e chain. Far t h e unsaturated acids t h e position a n d cis/trans n a t u r e of t h e double bond is indicated in parentheses. Table 2. Fatty Acid Methyl Ester Proportionality Factors (20) (DEGS Column a t 190)

F a t t y Acid Methyl Esters

24.17

A

Table 1. ECL Values for DEGS Column

Shorthand" notation (20)

18:O

Butter Margarine c o o k i n g oil

Figure 1. Graph for ECL number determination.

F a t t y acid m e t h y l ester

514:Oa 16:0

Proportionality Factors (PF)

myristic palmitic stearic oleic

the ECL value for the unsaturated ester (14-16). Our plot is shown in Figure 1. Table 1 gives the ECL values calculated from Figure 1 for a composite sample by our students and the corresponding literature values (17). The agreement is excellent. Once these ECL values have been determined, the components of a commercial fat or oil can be easily measured. The students can use our ECL values directly or, in a longer laboratory class, can determine their awn ECL values by the method outlmed above. Quantitation: Gas chromatographic detectors seldom show a direct relationship between peak area and sample component ratios and for this reason peak area proportionality factors are necessary (18). Rather than include this calibration as a part of this experiment we have relied on the published pmmztionalitv factors (20). The peak area for each fatty acid ester is calculated by triangulation (peak height multiplied by peak width at half height) and then multiplied by the proportionality factors (PF) found in

Cooking oil -~~~~ B Olive oil P e a n u t oil Safflower oil A Safflower oil B Salad oil A Salad oil B Sesame seed oil Shortening Bovine d e p o t fat Porcine depotfat

18:l

18:2

18:3

1.46 16.26

...

...

33.94 12.22

1 2 . 8 3 25.25 8.14 63.37

...

8.76

5 . 4 1 44.81

2.12 ...

17.86 14.06 10.27

2 . 7 3 22.48 54.79 3.83 67.03 15.07 1 . 7 6 47.28 38.04

...

5.42

1 . 1 9 10.40 82.98

... ... ...

6.50 10.60 10.38

... .. .

9.16 15.60

5.36 3 8 . 2 47.16 10.62 43.52 28.70

7.19

25.94

11.05 46.55

2.00

...

2'13

25.86

11.14 47.86

10.38

...

...

.. .

38.34 2 . 6 8

... ... ...

1 . 6 8 12.73 79.09 . . . 4 . 2 3 42.72 35.56 2.31 4.26 43.47 39.19 2.68 0.25 1.55

These values m a y he slightly low d u e t o t h e volatility of s a m e of t h e lower molecular weight esters. a

Table 2. The individual weight percentages are calculated from the total corrected area of all peaks excluding the internal standard (17:O). These P F values are for a thermal conductivity detector. If flame ionization is used different factors must be employed (19). More accurate results can be obtained if a recorder integrator is available.

The Experiment Students are either given samples of cooking fats and ails obtained from a supermarket, or asked to bring in their family's favorite product. The transesterification is performed; the methyl esters are injected into the gas chromatograph and a chromatogram obtained. This whole procedure takes 60-90 min. A sample student chromatogram of e commercial salad oil is shown in Figure 2. From the chromatogram the student identifies the sample eamponents by calculating the relative retention times (relative to 17:O) and from the logarithm of these relative retention times the ECL values are ohtained from Figure 1. The components are then identified fmm the ECL values in Table 1. Peak area measurements are then performed and quantitation is ohtained by applying the proportionality factors inTable 2. Representative results are shown in Table 3. They show very gmd agreement with published specifications (9). Our students have found it very interesting to compare various brands cf salad and cooking oils and to compare the "health food" oils to the name brand oils of the same material.

Literature Cited (1) ConsumrrRe~orts.38.553 119731. (21 James. A.T.. Martin. A. J. P.. and Smith, G. H..J. Blochem.. 52.262 (1952). (3) Wharton, D. C., and MeCarty. R. E.. "Experiments and Methods in Bi~ehemin-

Volume51. Number 6 , June 1974 / 407

M ~ ~CO..~N- ~ york, I IWZ: I 1ksn, ~ ~R.. - N ~ P ~~ ~ ~V C~ A~ C~~~ .. -I demic Prerr. Lac.. Now York, 1969. (4) Jamieson, G. R.. "Topics in Lipid Chemi~try." (Editor Gunatone, F. D.). Vol. 1. Wilcy-lnuracience. New York, 1910. pp 1W-160. (51 Stein, R. A . Slswson. R. A,. and Mead, J. F., "Lipid Chmmatographic Analysis." (Editor: Marinctti, G. V.), Vol 1, Marcel Dekker, Ine., New York. 1067, pp. 361-4W. (6) Gunsfone. F. D., "An Intraduetion to the Chemistry and Biochemistry of Fatty AcidaandTheirGlyceride%:2ndEd.. Chapman-Hall. London. 1967. (7) Chapman. D.. "lntmduction to Lipids." MeGrar.Hill Bwk Co.. London, 1969. (81 Litchfield. C.. "AnelysisofMglyee~des." Academic Press,Inc.. NmYork, 1972. I91 O'Connot. R. T.. and Herb. S. F.. J. Amer Oil Chsm. Soc., 47.186A 119701. (10) Forman, W.A., andPaulmn,D.R., J.CHEM.EDUC.,49,572119721. trill, ~h~

408

/

Journal of Chemical Education

i l l ] M ~ ~ C ~L.D., I B . a n d ~ c h m i t . , ~ . ~ . , ~cn ah ~m. 33.a63i19611. (12) Fioriti. J.A.. Burde. N.andSim8.R. J., J. Amar Oilcham. Soe., 46.108i19691. (13) Car-Chrom. Neurletter, W N o . liI9691. 1141 Bottcher. C. J. F.. Wmdford, F. P., Balsma-van Houfe. E., and van Gent. C. M.. Re