Laboratory 7 Lipid chemistry - fatty acids Introduction The lipid fraction of foods is very complex in that it is comprised of acylglycerols

(mono-, di- and triglycerides), phospholipids, and minor lipids including glycolipids, cholesterol and other sterols, fat soluble vitamins (A,D,E,K), axes, hydrocarbons, and some pigments! "onoglycerides and diglycerides occur 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!, soy lecithin)! 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 lipid classes present as ell as the fatty acid composition and distribution ithin each class! &tereospecific analysis of triglycerides provides very useful information because of the influence of fatty acid stereospecific arrangement on the physical properties of the fat! 'or many purposes, simple chemical measurements 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 the iodine and fatty acid composition A) Gas chromatography of fatty acid methyl esters 'atty acid ('A) composition affects oxidative stability, physical properties and nutritional aspects of lipids! (ils are categori%ed depending on the 'A composition) linolenic (soy, canola), linoleic (corn, cottonseed, safflo er, sunflo er), oleic (olive oil, high oleic varieties), lauric (palm *ernel, coconut), etc! +y far the most common vegetable oil sold in the ,& is soybean oil! -o ever, current interest in nutritional implications of different oils has stimulated gro th in sales of olive oil! "editerranean coo*ing (.talian, /ree*, &pain, &outhern 'rance) uses olive oil, in part for flavoring! +ecause olive oil is expensive, restaurants tend to use cheaper oils if they can! -o ever, authentic .talian food should be coo*ed using olive oil! 0ou ill analy%e the fatty acid compositions of several vegetable oils in today1s lab and 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 (54(T) capillary columns! These columns have the li3uid phase lining the inner all of an open tube from 6!7-6!8 mm internal diameter and 79-766 meters in length! This arrangement produces lo er bac* pressure and allo s longer columns to be used, compared to pac*ed columns! The pac*ed columns rely on a pac*ing of solid support that is coated ith a film of the li3uid phase! Due to the high bac* pressure, lengths are limited to :-; meter, drastically cutting separation efficiency compared to capillary columns! +ecause triglycerides, phospholipids and most free fatty acids are not volatile enough to be chromatographed 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 for free fatty acids and less so for triglycerides (T/) or phospholipids (#<)! +asic catalysts are very rapid for T/ or #<, but inefficient for free fatty acids! .n this exercise, because the lipid is assumed to be predominantly acyl, basic catalysis using potassium methoxide is used! .n some cases here plasmalogens are present, acid cataly%ed transesterification causes dimethyl acetal artifacts to be formed that interfere ith chromatography! This is not a problem ith base cataly%ed methods!

Preparation of fatty acid methyl esters: This method is based on "ax ell and "armer, <ipids, 7=>;, 7>)?9;!

NOT : This part !ill already be prepared for you and !ill be run on the G"# $ou !ill recei%e handouts either in lab or on the !eb & you !ill not do this section#
Each group ill ta*e one different sample from those provided! The oils are soybean, 4anola, corn oil, sunflo er, peanut, olive and (live /arden salad dressing! The class data !ill be pooled# Therefore' be sure to get the data from the other lab groups# All tubes and glass are (pipettes) in contact ith the samples should be rinsed ith methanol before use! 7! Around :6 mg lipid (7 drop) is added to a lea*-proof teflon lined conical tube@ eigh tube ith sample and dissolve lipid in :!6 m< iso-octane! :! 766 < of : A K(- (7!7 gB76 m<) in methanol is added and the tube vortexed for C6 seconds! ;! Allo tube to stand 9-76 min! and the lo er methanol phase is discarded! ?! 6!9 m< saturated ammonium acetate (a3!) is added, the tube vortexed, and the lo er a3ueous layer is discarded after 9-76 min! 9! Add 7!6 m< ater to the tube, vortex again, allo to stand, and discard the lo er layer! C! Add a small amount of sodium sulfate, sha*e ell, and let sit for 76 minutes! 8! The li3uid layer is carefully transferred to a clean tube avoiding transfer of the solid sodium sulfate! >! 7 < of this sample is in$ected onto a gas chromatograph operated using the follo ing conditions hich have been optimi%ed for fast analysis) column D+-::9 ;6 x !:9 mm i!d!, helium carrier linear flo velocity ;: cmBsec, flame ioni%ation detector, split in$ector ratio >6)7, column oven temperature ::64, in$ectorBdetector ;664! 4hromatograms of fatty acid standards and samples ill be provided! "alculations and discussion 7D .dentify the fatty acids in the samples from retention values of *no n fatty acid standards! :D 4alculate the E fatty acid composition for each oil! Data ill be pooled! +e sure to have copies of all data for your lab report! () Iodine %alue The iodine value is a simple measure of the degree of unsaturation in a lipid sample! The iodine value is defined as the amount (g) of halogen (expressed as .) that combines ith 766 g of fat! This procedure is 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 test for unsaturation in an un*no n sample is to loo* for decolori%ation of +r : in 44l? hen it is added to your sample! 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 the sample, b) reduction of the remaining halogen to . :, c) iodometric measurement of .: ith sodium thiosulfate (Aa:&:(;)! "aterials - 5i$s solution (7C!9 g .4l in 7 < of glacial acetic acid) - potassium iodide 79E Bv a3ueous - sodium thiosulfate (6!7 ")

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

Group ) 7 : ; ? 9 C 8 > = 76 #rocedures

Lipids lab 7 &oy bean oil 4anola oil 4orn oil &unflo er oil #eanut oil Extra virgin oil (live garden oil ,n*no n 7) 4anola ,n*no n :) &unflo er ,n*no n ;) #eanut oil

Lipids lab * &oy bean oil 4anola oil 4orn oil &unflo er oil #eanut oil Extra virgin oil <ard "argarine &esame oil 4od liver oil

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/ PIP TT 6666 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< chloroform solution into duplicate :96 m< Erlenmeyer iodine flas*s! "a*e sure that the samples are completely dissolved! Also prepare a blan* containing only 76 m< of chloroform! :! Transfer :9 m< of 5i$s solution to each flas*! ;! &topper the flas*s and gently sha*e to mix! Allo to stand in the dar* for ;6 minutes! ?! 4arefully remove the stopper, add :6 m< of 79E K.! Add 766 m< of distilled ater, pouring so that the sides of the flas* are ell rinsed! Do not orry that there are t o phases, the 3uantification is based on the measurement of .: in the a3ueous phase! 9! Titrate ith 6!7 " sodium thiosulfate to a pale yello color! Add : m< of the starch suspension (the mixture should turn blue if you haven1t over titrated ith the thiosulfate)! Ao carefully continue the titration until the blue color $ust disappears! $ou may !ish to set7up your lab noteboo8 page !ith the follo!ing headings for data entry# FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF Gegetable &ample +lan* &ample 7 &ample : (ils 5gt (m<) (m<) (m<) .G 7 .G : Average FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF C! 4alculations) .odine Galue (.G) (m<b H m<s) (6!7 e3B7666 m<) (7:C g .Be3) (766 g) .G I ----------------------------------------------------------------------5gt of sample (g) here, m<b is the m<Js of blan*@ and m<s is the m<Js of sample Place bea8ers containing 9:; g each of peanut oil' soybean oil' butter' sunflo!er oil' oli%e oil' corn oil' and "anola into the <;" o%en for use in ne=t !ee8>s lab# 4alculations and discussion 7D All chromatograms ill be given! The students have to determine the identity of olive garden oil and un*no ns 7, : and ;! :D .n your report) 7! 4alculate the iodine value for all fat samples! :! 'or your groupJs sample only, calculate the iodine value from the chromatogram provided! ;! Examine the pooled class data (for fatty acids and iodine values) and relate them to the physical nature of the samples (hardness, li3uidity, etc!) and the origin of the samples! ?

Discuss the usefulness and limitations of these methods of lipid analysis! Kuestions for discussion and conclusion 7! 5hat oil appears to be used for the salad dressing in the (live /ardenL Mustify your ans er! :! 4ompare your .G values to *no n values for these fats!