in humans. Lipids include certain soluble vitamins such as A, D, and K. The scope of lipids is huge, and includes several ot her subgroups, including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids and polyketides (derived from condensation of ketoacyl subunits); and sterol lipids and preno l lipids. Based 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 back bone. Triacylglycerides have three fatty acids ester ified to the three OHs on glycerol. Glycerophospholipids have two fatty acids esterified at carbons 1 and 2, and a phospho-X groups esterifed at C3. The structure of these molecules determines their function. For example, the very insoluble triacylglycerides are used as the predominant storage form of chemical energy in the body. In contrast to polysaccharides such as glycogen (a polymer of glucose), the Cs in the ac yl-chains of the triacylglyceride are in a highly reduced state. Hence, lipids are considered to be more effective in long-term energy storage.
Methodology
GREASE-SPOT TEST y
Vegetable oil
y
Lecithin (.01g/mL DCM)
y
DCM
y
Filter paper
Four areas of a piece of filter paper were labeled as ³vegetable oil´, ³lecithin´, ³H2O´, and ³DCM´. Using Pasteur pipets, a drop of the appropriate substance was applied to its corresponding area. The filter paper was then warmed either by placing directly on a hot plate or placed on a watch glass over a boiling water bath. A clay triangle may be placed on top of the filter paper to keep heat distributed evenly. The results were then recorded.
SAPONIFICATION TEST
y
Vegetable oil
y
3M NaOH
Two large-sized test tubes were labeled ³oil´ and ³H2O´. 8 drops of oil and 8 drops of water were placed in their respective test tubes. 10 drops of 3M NaOH solution were p laced in each test tube. The tubes were p laced in a boiling water bath for approximately 15-20mins. After, the tubes were removed and allowed to cool to room temperature. 5mL of distilled water was then added to each tube, stoppered, and shaken vigorously. Observations at this stage were then recorded. The solutions in each test tube were acidified using a few drops of concentrated H2SO4, 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. A piece of red and blue litmus paper were dipped onto this material and acidity or basicity were noted. Observations were then recorded.
UNSATURATION TEST y
Vegetable oil
y
Glycerol
y
DCM
y
5% bromine-DCM solution
y
50mL buret
Two large-sized test tubes were labeled with ³o il´ and ³glycerol´. 3mL of dichloromethane (DCM) were placed in each tube. 10 drops each of vegetable oil and glycerol were placed in each test tube. The contents were thoroughly mixed. Under a fume hood, a funnel was used to add 5% bromine-dichloromethane solution to a 50mL buret. The initial volume was recorded. To each tube, the bromine-DCM solution was added dr op-wise until the reddish-brown color first appears. The final volume of the bromine-DCM solution was recorded and measured.
Results and Discussion Table 1. Results of the 3 tests on various lipid samples
Sample
Grease spot
Saponification Solution
Unsaturation
Oily residue
(drops)
Margarine
With grease spot
Acid
Acid
17
Baguio oil
With grease spot
Acid
Acid
11
Canola oil
With grease spot
Acid
Acid
60
Sesame oil
With grease spot
Acid
Acid
56
Palm oil
With grease spot
Acid
Acid
12
Olive oil
With grease spot
Acid
Acid
48
Fish oil
With grease spot
Acid
Acid
15
Pork fat
With grease spot
Acid
Acid
37
Lard
With grease spot
Acid
Acid
50
Butter
With grease spot
Acid
Acid
30
The grease spot test aims to det ermine whether or not a lipid contains a glycerol or sphingosine (backbone component for sphingolipids). Howver, the sample to be tested must be in liquid form for it to be tested. Semi-solid lipids must first be melted before subjected to the grease-spot test. Lipids derived from either glycerol or sphingosine will produce a t ranslucent µgrease spot¶ on the filter paper, while other lipids from derived from these two will not.
Fig. 1 Sphingosine
Fig. 2 Glycerol
As shown in the table, all of the samples tested positive for
the
grease-spot test, having left a translucent spot on the filter
paper. It
can be concluded, therefore, that all the samples are either derived from sphingosine or glycerol.
Saponification is a process where lipids with fatty ac id ester linkages that undergo hydrolysis, catalyzed by a strong base or acid. Hydrolysis through a base is called saponification and is the primitive method for producing lye-soap. The p rocess of which is to boil animal fat in a container with lye (a strong base) then skimming off the r esidue that forms on top. As with the grease-spot test, all the samples formed a residue on to p of the solution. It can be co ncluded that all the samples contain fatty acid ester linkages that hydrolyze.
Fig. 3 Formation of soap
For lipids with C-C double bonds, add ition reactions will occur. This happens when additional atoms ³add´ to the C-C double bond which results in its saturation. Oils generally have more sites for saturation; and by synthetic addition of hydro gen to saturate the oil, hydrogenation occurs. It changes the o il into a fat. In 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. Given the results in tab le 1, Canola oil is shown to be the least saturated (60 drops) and Bagiuo oil is shown to be the most saturated (11 drops).
References rd
Boyer, R. (2006). ³Concepts in Biochemistry´. John Wiley & Sons (Asia). 3 ed. Heinz E.(1996). Plant glycolipids: structure, isolation and analysis. Advances in Lipid Method ology - 3, pp. 211±332
Menguito, C., et al. (2010). ³Basic Laboratory Experiments in Biochemistry´. College of Science, University of Santo Tomas. Michelle A, Hopkins J, McLaughlin CW, Johnson S, Warner MQ, LaHart D, Wright JD (1993). Human Biology and H ealth. Englewood Cliffs, New Jersey, USA: Prentice Hall.
