CHARACTERIZATION CHARACTERIZATION OF SAPONIFIABLE LIPIDS
Abstract The objective of the following series of experiments is to attempt to characterize saponifiable lipids. These are otherwise known as simple lipids and can be hydrolyzed under basic conditions. The series of experiments include the grease-spot test which uses a filter paper as medium on which to check for the ‘spot’ simple lipids make. The next experiment is called the saponification test wherein the ability of a lipid to hydrolyze in the presence of a strong acid in this case! "# $a%&'. The final test would would be the unsaturation test! which involves a similar method to titration! titration! but using () bromine-*+# bromine-*+# on the lipid.
,ipid - a substance such as a fat! oil or wax that dissolves in alcohol but not in water. ,ipids contain carbon! hydrogen and oxygen but have far less oxygen proportionally than carbohydrates. aponifiable - the hydrolysis of an ester under b asic conditions to form an alcohol and the salt of a carboxylic acid carboxylates'. nsaturated - a fat or fatty acid in which there are one or more double bonds in the fatty acid chain
,ipids are a broad group of biochemical compounds that! along with carbohydrates and proteins! make up the majority of our diet. ,ipids are relatively harder to characterize since they have no functional groups that are uni/ue to them! as compared to proteins with amino acids and carbohydrates with aldehyde or ketone forms of alcohol groups. 0enerally! lipids are insoluble in water! but are soluble in other organic solvents such as chloroform and methanol. They form globules in water which may be in the form of a liposome! micelle! or a bilayer sheet characterizing the cell membrane'. ,ipids are present in our body primarily as energy storage media. 1side 1side from this! they also function as insulators to regulate body heat2 hence! they are found just below the epidermal layer
in humans. ,ipids include certain soluble vitamins such as 1! *! and 3. The scope of lipids is huge! and includes several other subgroups! including fatty acyls! glycerolipids! glycerophospholipids! sphingolipids! saccharolipids and polyketides derived from condensation of ketoacyl subunits'2 and sterol lipids and prenol lipids. 4ased on their reaction with strong bases! however! lipids can be divided into two major groups! the saponifiable and nonsaponifiable lipids. The major saponifiable lipids are triacylglycerides! glycerophospholipids! and the sphingolipids. The former two use glycerol as the backbone. Triacylglycerides have three fatty acids esterified to the three %&s on glycerol. 0lycerophospholipids have two fatty acids esterified at carbons 5 and 6! and a phospho-7 groups esterifed at +". The structure of these molecules determines their function. 8or example! the very insoluble triacylglycerides are used as the predominant storage form of chemical energy in the body. 9n contrast to polysaccharides such as glycogen a polymer of glucose'! the +s in the ac yl-chains of the triacylglyceride are in a highly reduced state. &ence! lipids are considered to be more effective in long-term energy storage.
Metodo!o"# 0:;1;-5g?m, *+#' *+# 8ilter paper
8our areas of a piece of filter paper were labeled as @vegetable oilA! @lecithinA! @&6%A! and @*+#A. sing mins. 1fter! the tubes were removed and allowed to cool to room temperature. (m, of distilled water was then added to each tube! stoppered! and shaken vigorously. %bservations at this stage were then recorded. The solutions in each test tube were acidified using a few drops of concentrated &6%C! and checked with blue litmus paper. The solutions were mixed with a stirring rod. The material formed on top of the solution was noted for each tube. 1 piece of red and blue litmus paper were dipped onto this material and acidity or basicity were noted. %bservations were then recorded.
Two large-sized test tubes were labeled with @oilA and @glycerolA. "m, of dichloromethane *+#' were placed in each tube. 5> drops each of vegetable oil and glycerol were placed in each test tube. The contents were thoroughly mixed. nder a fume hood! a funnel was used to add () bromine-dichloromethane solution to a (>m, buret. The initial volume was recorded. To each tube! the bromine-*+# solution was added drop-wise until the reddish-brown color first appears. The final volume of the bromine-*+# solution was recorded and measured.
Resu!ts and Discussion Tab!e $% :esults of the " tests on various lipid samples
0rease spot Dith grease spot
aponification olution %ily residue 1cid
nsaturation drops' 5E
4aguio oil +anola oil esame oil
The grease spot test aims to determine whether or not a lipid contains a glycerol or sphingosine backbone component for sphingolipids'. &owver! the sample to be tested must be in li/uid form for it to be tested. emi-solid lipids must first be melted before subjected to the grease-spot test. ,ipids derived from either glycerol or sphingosine will produce a translucent ‘grease spot’ on the filter paper! while other lipids from derived from these two will not.
Fig. 1 Sphingosine
Fig. 2 Glycerol
1s shown in the table! all of the samples tested positive for the grease-spot test! having left a translucent spot on the filter paper. 9t can be concluded! therefore! that all the samples are either derived from sphingosine or glycerol. aponification is a process where lipids with fatty acid ester linkages that un dergo hydrolysis! catalyzed by a strong base or acid. &ydrolysis through a base is called sapon ification and is the primitive method for producing lye-soap. The process of
to boil animal fat in a container with lye a strong base'
skimming off the residue that forms on top. 1s with the
spot test! all the samples formed a residue on top of the solution. 9t can be concluded that all the samples contain fatty acid ester linkages that hydrolyze.
Fig. 3 Formation of soap
8or lipids with +-+ double bonds! addition reactions will occu r. This happens when additional atoms @addA to the +-+ double bond which results in its saturation. %ils generally have more sites for saturation2 and by synthetic addition of hydrogen to saturate the oil! hydrogenation occurs. 9t changes the oil into a fat. 9n the unsaturation test! how many drops until a sample is saturated by bromine can give an approximate scale to which to base how unsaturated a sample is. The more drops it takes to saturate appearance of brown color' a sample! the more unsaturated a sample is. 0iven the results in table 5! +anola oil is shown to be the least saturated F> drops' and 4agiuo oil is shown to be the most saturated 55 drops'.
4oyer! :. 6>>F'. @+oncepts in 4iochemistryA. Gohn Diley H ons 1sia'. "rd ed. &einz ;.5IIF'. 5>'. @4asic ,aboratory ;xperiments in 4iochemistryA. +ollege of cience! niversity of anto Tomas. #ichelle 1! &opkins G! #c,aughlin +D! Gohnson ! Darner #L! ,a&art *! Dright G* 5II"'. Hman !iology and Health. ;nglewood +liffs! $ew Gersey! 1J