Digitally signed by Jason Raquin Roque DN: cn=Jason Raquin Roque, o, ou, email=jason_mike15@yahoo. com, c=PH Date: 2012.05.

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Reaction of Lipids
Roque, Jason R. Hernandez, Ritz Hendrie C. Frias, Abigail Pauline F. Someros, Kristine Carl S. Bachelor of Science in Biology Major in Human Biology College of Science De La Salle University Dasmarias Dasmarias, Cavite, Philippines

ABSTRACT

The experiment is all about the reactions of lipids in different tests. The lipid samples that were used in this experiment are: egg yolk, butter, coconut oil, olive oil, cod liver oil, lecithin and cholesterol. The first test was the Iodine test; each sample was prepared with chloroform and iodine solution was added into it drop by drop until initial discoloration was observed. All samples reacted by only needing 1-3 iodine drops to display changes except for olive oil which required 5 drops of iodine. It was deduced that coconut oil and butter are unsaturated while olive oil, cod liver oil, cholesterol, lecithin and egg yolk are saturated lipids because of the presence of C=C double bond. Next is the Acrolein test, a test for the presence of glycrol; each sample had KHSO4 added into it and had been subjected in a water bath to test for the smell each produces. All samples smelled like burnt grease except for butter and cod liver oil which smelled like burnt butter grease and fish smell respectively. In Saponification, each sample was prepared with chloroform and KOH and was subjected in a water bath. Cold, saturated NaCl solution was then added and the formation of soap precipitate was observed. Only the egg yolk and the cholesterol showed no formation of soap precipitate while the rest did. Lastly in Liebermann-Burchard test, each sample was prepared with chloroform, acetic anhydride, and concentrated H2SO4. Change in color was observed; all the sample solutions showed positive result by displaying a blue-green color except for olive oil, cod liver oil and lecithin. The qualitative analysis of lipids through various reactions was done to characterize or identify the lipid samples used. Each of the lipids has their own characteristics which made them unique.

INTRODUCTION Lipids are very diverse in both their respective structures and functions. These diverse compounds that make up the lipid family are so grouped because they are insoluble in water. They are however soluble in other organic solvents such as ether, acetone and other lipids. Major lipid groups include fats, phospholipids, steroids and waxes.2 This definition has echoes of Bloors "simple and compound lipoids". In practice, it is often necessary to subdivide the main groups further. For example, the complex lipids for many purposes are best considered in terms of either the glycerophospholipids (or simply as phospholipids), which contain a polar phosphorus moiety and a glycerol backbone, or the glycolipids, which contain a polar carbohydrate moiety.2 For many years, lipids were considered to be intractable and uninteresting oily materials with two main functions to serve as a source of energy and as the building blocks of membranes.3 They were certainly not considered to be appropriate candidates for such important molecular tasks as intracellular

signalling or local hormonal regulation. In 1929, George and Mildred Burr demonstrated that linoleic acid was an essential dietary constituent, but it was many years before the importance of this finding was recognized by biochemists in general. With the discovery by Bergstrm, Samuelsson and others in 1964 that the essential fatty acid arachidonate was the biosynthetic precursor of the prostaglandins with their effects on inflammation and other disease states, the scientific world in general began to realize that lipids were much more interesting than they had previously thought.2 A major milestone was achieved in 1979 with the discovery of the first biologically active phospholipid, platelet-activating factor. At about the same time, there arose an awareness of the distinctive functions of phosphatidylinositol and its metabolites. Since then, virtually every individual lipid class has been found to have some unique biological role that is distinct from its function as a source of energy or as a simple construction unit of a membrane. Indeed it is now recognised that lipids in membranes function also in the trafficking of cellular constituents, the regulation of the activities of membrane proteins and signalling. All multi-cellular organisms, use chemical messengers to send information between organelles and to other cells and as relatively small hydrophobic molecules, lipids are excellent candidates for signalling purposes.4 The fatty acid constituents have well-defined structural features, such as cis-double bonds in particular positions, which can carry information by binding selectively to specific receptors. In esterified form, they can infiltrate membranes or be translocated across them to carry signals to other cells. During transport, they are usually bound to proteins so their effective solution concentrations are very low, and they are can be considered to be inactive until they reach the site of action and encounter the appropriate receptor. Storage lipids, such as triacylglycerols, in their cellular context are inert, and indeed esterification with fatty acids may be a method of de-activating steroidal hormones, for example, until they are actually required. In contrast, polar phospholipids have both hydrophobic and hydrophilic sites that can bind via various mechanisms to membrane proteins and influence their activities. Glycosphingolipids carry complex carbohydrate moieties that have a part to play in the immune system, for example. Lipids have been implicated in a number of human disease states, including cancer and cardiovascular disease, sometimes in a detrimental and sometimes in a beneficial manner. In short, every scientist should now be aware that lipids are just as fascinating as all the other groups of organic compound that make up living systems.2

MATERIALS / REAGENTS & EXPERIMENTAL PROCEDURE The reaction of butter, egg yolk, coconut oil, olive oil, cod liver oil, lecithin, and cholesterol was observed by conducting four types of test. First is the Iodine Test, a pinch of butter and drops of egg yolk, coconut oil, olive oil and cod liver oil was placed in separate test tube and was dissolved in 1.00 ml chloroform. Iodine solution was added on the samples drop by drop until the discoloration was observed. Same as well in 1.00 ml lecithin and cholesterol solution, it was placed on separate test tube and iodine solution was also added. Second is the Acrolein Test, same sample was used as in the first test. A pinch of KHSO4 was added on the samples, and then it was heated for some time. After heating it, the smell of each sample was recorded. Third is the Saponification, same sample was used this time 2.00 ml of ethanolic KOH was added then it was heated for 5 minutes. 5.00 ml of cold saturated NaCl solution was added and formation of soap precipitates was observed. Last is the Lieberman-Burchard Test, still the same sample was used 5 drops of acetic anhydride and 2 drops of concentrated H2SO4 was added to each sample then the changed in color was observed.

DATA & RESULTS The lipids are a large and diverse group of naturally occurring organic compounds that are related by their solubility in nonpolar organic solvents (e.g. ether, chloroform, acetone & benzene) and general insolubility in water. The tables that will be shown and discussed below will give a better understanding of how lipids react with other chemicals Table 1. Test for Unsaturation SAMPLES Butter Egg yolk Coconut oil Olive oil Cooked Pork Fat Lecithin Cholesterol # of Drops of Iodine 20 35(no changes) 5 5 5 1 8

The test for Unsaturation of lipids is used to test for the presence of starch. Iodine solution iodine dissolved in an aqueous solution of potassium iodide reacts with starch producing a purple black color. The colour can be detected visually with concentrations of iodine as low as 0.00002M at 20C. However the intensity of the colour decreases with increasing temperature and with the presence of water-miscible, organic solvents such as ethanol. Also the test cannot be done at very low pHs due to the hydrolysis of the starch under these conditions. This test identifies the level of saturation and the number of bonds an oil, fat or lipid has. The more unsaturated, multi-bonded, the lipid is, the more it absorbs iodine. The less iodine it absorbs, the lipid is considered to be saturated, single bonded. The typical iodine test drops for the samples should have been in the range of this table below. Table 2. Typical Iodine #'s Coconut oil Butter Beef tallow Palm oil Lard Olive oil 8 - 10 25 - 40 30 - 45 37 - 54 45 - 70 75 - 95

Peanut oil Cottonseed oil Corn oil Fish oils Soybean oil Safflower oil Sunflower oil Linseed oil

85 - 100 100 - 117 115 - 130 120 - 180 125 - 140 130 - 140 130 - 145 170 - 205

Acrolein (systematic name: propenal) is the simplest unsaturated aldehyde. It is produced widely but is most often immediately reacted with other products due to its instability and toxicity. It has a piercing, disagreeable, acrid smell, smell of burning fat, which is caused by glycerine in fat decomposing. Acrolein is a compound formed by dehydration of glycerol, so its presence indicates the presence of a glyceride ester (usually a triglyceride) Example: a fat or oil. The smell is a bit like a barbecue. Acrolein test is a test for the presence of glycerin or fats. A sample is heated with potassium bisulfate, and acrolein is released if the test is positive. When a fat is heated strongly in the presence of a dehydrating agent such as KHSO4, the glycerol portion of the molecule is dehydrated to form the unsaturated aldehyde, acrolein (CH2=CH-CHO), which has the peculiar odor of burnt grease. Table 2. Acrolein Test SAMPLES Butter Egg yolk Coconut oil Olive oil Cod liver oil Lecithin Cholesterol Characteristic Smell Burnt butter Burnt grease Burnt grease Burnt grease Fishy smell Burnt grease Minty-like smell

The principle behind the acrolein test is a specific chemical reaction. This reaction is utilized to determine the presence of glycerin in a fat. By heating the fat sample in the presence of potassium bisulfate (KHSO4), which acts as a dehydrating agent, acrolein (C3H4O, or CH2=CH-CHO) is formed and can easily be detected by its odor. Whenever fat is heated in the presence of a dehydrating agent, the fat molecule will shed its glycerol in the form of the unsaturated aldehyde - acrolein.

Acrolein smells like burned grease, and this toxic chemical was used in the first World War as a chemical weapon. Even in small concentrations, exposure irritates the mucous membranes and causes the eyes to tear up. It can incapacitate individuals very quickly at levels of only a few parts per million. Saponification of oils means the hydrolysis of triglycerides in the oil in presence of an alkaline medium (e.g. NaOH) into glycerol and fatty acids with the production of sodium salt of fatty acids or soap. A test for unsaturated steroids (as cholesterol) and triterpenes based on the formation of a series of colors (as pink to blue to green) with acetic anhydride in the presence of concentrated sulfuric acid called the Liebermann Burchard test. Table 3. Saponification and Liebermann-burchard Test SAMPLES Butter Egg yolk Coconut oil Olive oil Cod liver oil Saponification Thick yellow soapy layer on top White precipitate forms Brown precipitate forms Thin white layer on top Cloudy white solution w/ white precipitate Opaque white w/ thick layer bubble on top White precipitate Liebermann-burchard Light pink (-) Blue green (+) Clear yellow (-) Blue green (+) Black (-)

Lecithin Cholesterol

Brown (-) Blue green (+)

Saponification Number the number of milligrams of alkali required to neutralize the fatty acids contained in one gram of fat is used as an indicator of fatty acid chain length in triacylglycerols and triacylglycerols containing long fatty acids have lower saponification number than those with shorter fatty acids Liebermann Burchard is a test for the presence of sterol. emerald green color indicates positivity to the test. Formation of a green or green-blue color after a few minutes is positive. Ideally, the cholesterol solution gives a nice reaction, the coconut oil should show no significant color change, and the lard gives a weak reaction. The results for this test have been variable over the past few years. For example, coconut oil sometimes gives a green color, and unsaturated oils invariably do, whereas lard often gives a brown color. I think the variability has been due to using old solutions; they should be made fresh with reasonably fresh samples, which is fairly inconvenient.

REFERENCE 1. 2. 3. 4. Legaspi, G. A. 2009. Essentials of Biochemistry Laboratory Retrieved from http://biology.about.com/od/molecularbiology/ss/lipids.htm on August 22, 2011 Retrieved from http://lipidlibrary.aocs.org/Lipids/Nomen/index.htm on August 22, 2011 Retrieved from http://lipidlibrary.aocs.org/Lipids/whatdo/index.htm on August 22, 2011