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Laboratory 7

Lipid chemistry - fatty acids

Introduction

The lipid fraction of foods is very complex in that it is comprised of acylglycerols (mono-, di- andtriglycerides), phospholipids, and minor lipids including glycolipids, cholesterol and other sterols, fatsoluble vitamins (A,D,E,K), axes, hydrocarbons, and some pigments! "onoglycerides and diglyceridesoccur in some foods as hydrolysis products of triglycerides or as additives used for their surface activity!#hospholipids, the ma$or class of natural polar lipid, are also used in certain foods as emulsifiers (i!e!, soylecithin)! The texture, flavor, viscosity, and emulsion behavior are influenced by the lipids in foods!

A thorough characteri%ation of lipids ould include chromatographic analysis of the different lipidclasses present as ell as the fatty acid composition and distribution ithin each class! &tereospecificanalysis of triglycerides provides very useful information because of the influence of fatty acidstereospecific arrangement on the physical properties of the fat! 'or many purposes, simple chemicalmeasurements provide sufficient data for general characteri%ation of a lipid sample!

This exercise ill consist of chemical characteri%ation of selected pure fats and oils by examining theiodine and fatty acid composition

A) Gas chromatography of fatty acid methyl esters

'atty acid ('A) composition affects oxidative stability, physical properties and nutritional aspects oflipids! ils are categori%ed depending on the 'A composition linolenic (soy, canola), linoleic (corn,cottonseed, saffloer, sunfloer), oleic (olive oil, high oleic varieties), lauric (palm *ernel, coconut), etc!+y far the most common vegetable oil sold in the & is soybean oil! oever, current interest innutritional implications of different oils has stimulated groth in sales of olive oil! "editerranean coo*ing(.talian, /ree*, &pain, &outhern 'rance) uses olive oil, in part for flavoring! +ecause olive oil isexpensive, restaurants tend to use cheaper oils if they can! oever, authentic .talian food should be

coo*ed using olive oil! 0ou ill analy%e the fatty acid compositions of several vegetable oils in today1s laband determine the identity of an oil used for salad in a local .talian 2estaurant, the live /arden!

'atty acid identification and 3uantification is most accurately done using gas chromatography (/4)using polar all coated open tubular (54T) capillary columns! These columns have the li3uid phaselining the inner all of an open tube from 6!7-6!8 mm internal diameter and 79-766 meters in length! Thisarrangement produces loer bac* pressure and allos longer columns to be used, compared to pac*edcolumns! The pac*ed columns rely on a pac*ing of solid support that is coated ith a film of the li3uidphase! Due to the high bac* pressure, lengths are limited to :-; meter, drastically cutting separationefficiency compared to capillary columns!

+ecause triglycerides, phospholipids and most free fatty acids are not volatile enough to bechromatographed directly, they must first be converted into methyl esters! This can be affected by using

acidic or basic catalysts, each ith advantages and disadvantages! Acidic catalysts are very rapid forfree fatty acids and less so for triglycerides (T/) or phospholipids (#

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Preparation of fatty acid methyl esters:This method is based on "axell and "armer, ;, 7>?9;!

NOT: This part !ill already be prepared for you and !ill be run on the G"# $ou

!ill recei%e handouts either in lab or on the !eb & you !ill not do this section#

Each group ill ta*e one different sample from those provided! The oils are soybean, 4anola, corn oil,sunfloer, peanut, olive and live /arden salad dressing! The class data !ill be pooled# Therefore'be sure to get the data from the other lab groups#

All tubes and glassare (pipettes) in contact ith the samples should be rinsed ith methanol before use!

7! Around :6 mg lipid (7 drop) is added to a lea*-proof teflon lined conical tube@ eigh tube ith sampleand dissolve lipid in :!6 m< iso-octane!

:! 766 < of : K (7!7 gB76 m! 7 < of this sample is in$ected onto a gas chromatograph operated using the folloing conditions hichhave been optimi%ed for fast analysis column D+-::9 ;6 x !:9 mm i!d!, helium carrier linear flovelocity ;: cmBsec, flame ioni%ation detector, split in$ector ratio >67, column oven temperature::64, in$ectorBdetector ;664! 4hromatograms of fatty acid standards and samples ill beprovided!

"alculations and discussion

7 .dentify the fatty acids in the samples from retention values of *non fatty acid standards!

: 4alculate the fatty acid composition for each oil! Data ill be pooled! +e sure to have copies of alldata for your lab report!

() Iodine %alue

The iodine value is a simple measure of the degree of unsaturation in a lipid sample! The iodine valueis defined as the amount (g) of halogen (expressed as .) that combines ith 766 g of fat! This procedureis based on the ability of unsaturated organic compounds to undergo addition reactions ith halogens(mainly iodine)! A mixed halogen molecule, typically .4l (iodine monochloride) or*s best! A classic testfor unsaturation in an un*non sample is to loo* for decolori%ation of +r:in 44l?hen it is added to yoursample! The loss of color is due to loss of +r:hich adds directly to the unsaturated carbons!

The general procedure for determining of iodine value involves a) addition of excess halogen to thesample, b) reduction of the remaining halogen to .:, c) iodometric measurement of .:ith sodiumthiosulfate (a:&:;)!

"aterials

- 5i$s solution (7C!9 g .4l in 7 < of glacial acetic acid)- potassium iodide 79 Bv a3ueous- sodium thiosulfate (6!7 ")

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- soluble starch 6!9 in distilled ater boiled to solubili%e the starch- chloroform- lipid samples:

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Group Lipids lab 7 Lipids lab *

7 &oy bean oil &oy bean oil: 4anola oil 4anola oil; 4orn oil 4orn oil? &unfloer oil &unfloer oil9 #eanut oil #eanut oil

C Extra virgin oil Extra virgin oil8 live garden oil n*non 7 4anola "argarine= n*non : &unfloer &esame oil76 n*non ; #eanut oil 4od liver oil

#rocedures

ALL P+O",-+. A+ TO ( ,ON NTI+L$ IN T/ /OO, -.ING GLO0.AN, GOGGL. ("A-. O1 T/ TO2I"IT$ O1 T/ "/3I"AL. T/AT 4ILL (-.,# 5,O NOT 3O-T/ PIPTT6666 T/. "/3I"AL. A+ ,ANG+O-.)

7! #repare a solution containing :6 mg of lipids per 7 m< chloroform! Transfer 76 m< of a :6 mg lipidBm