Experiment #6

Characterizati Charac terization on of Lipids of Melted M elted Fat, Lecithin, Glycerol and Vegetable Oils through Qualit Qua litative ative Tests *Pua Phee, Marie Pamela Celestine E. Relopez, Maria Charmella P. San Pedro, Anna Paula R. So, Ignacius Ignacius de Charles T. T. College of Science, Univ University ersity of Santo Tomas, Tomas, Espa E spa ña Blvd., Manila

Abstract Lipids are a diverse diverse group of molecules t hat are are hydrophobic and soluble in non-polar solvents. They function function as the major c onstituents of biological biological membranes. membranes. Lipids c an be class ified as saponifiable saponifiable or nonsaponifiable. Saponifiable Saponifiable lipids are esters of fatty acids that can under undergo go saponification (base hydrolysis of the esters of fats or oils to affor afford d glycerol and t he salt of the corre c orresponding sponding fatty acid . This This experiment c haracterizes haracterizes saponifiable saponifiable lipids through through four four tests . Grease-spot Grease-spot test t est indicates the presence of lipids; a filter paper was used and results show t hat the vegetable vegetable oil sample and leci tihin are positive positive (translucent spot). Saponification test detects whether the sample has ester bonds bonds . Hydrolysi Hydrolysis s and neutralization of the fatty acids through addition of NaOH and dehydration and acidification through adding H₂SO₄ yielded bubbles and a precipitate, and an acidic pH tested by a litmus paper if the sample is positiv positi ve. The vegetable vegetable oil and melted melt ed fat tested test ed positiv posit ive, e, while whil e the H₂O tested negativ negative. e. Acrolein test detects the presence of glycerols. Upon the addition of KHSO ₄, a dehydrating agent, a pungent odor and blackening of the mix ture indicates indicates a positive positive result. Unsaturation test determines t he degre degree e of saturation and the presence of double bonds. The The reagent bromine-dic bromi ne-dichlorometha hlorometha ne binds to the double bonds of the unsaturated unsaturated fatty acids which means that the vegetable vegetable oil, melted fat, and and glycerol glyc erol tests as positive.

Introduction

Biological lipids are a chemically diverse group of molecules, the common and defining feature of which is their insolubility in water. They are generally hydrophobic. Their functions vary as stored forms of energy in many organisms, phospholipids and sterols are major structural elements of biological membranes. Other lipids, although present in relatively small quantities, play crucial roles as enzyme cofactors, electron 1

carriers, light-absorbing pigments, hydrophobic anchors for proteins, “chaperones” to help membrane proteins fold, and emulsifying agents in the digestive tract, hormones, and intracellular messengers (Nelson & Cox, 2005). Lipids are non-polar, making them soluble in non-polar solvents (like dissolves like). The simplest lipids are the fatty acids, which constitute many complex lipids. They can be further divided to saturated and unsaturated fatty acids. Saturated fatty acids have carbons that are saturated with H atoms and only have single bonds, while unsaturated fatty acids contain one or more double bonds. Many important naturally occurring fatty acids aci ds are unsaturated unsaturated (Appling, (App ling, AnthonyAnthony-Cahi Cahill, ll, & Matthews, 2016). 20 16). Fats or triacylglycerols are the form of storage of fatty acids in organisms, they are triesters of fatty acids and glycerol. Those triacylglycerols that are solid are called fats, while those that are liquid are called oils. Phospholipids are lipids with phosphate-containing groups. As stated, lipids are major components of biological membranes, and the major classes include the glycerophosph glycerophospholipid olipids, s, sphingolipid sphingolipids s and gly g lycosphingol cosphingolipi ipids ds (in animals). Glycerolipids Glycerolipids are widespread in plant and bacterial membranes (Appling, Anthony-Cahill, & Matthews, 2016). Lipids can also be classified as saponifiable or nonsaponifiable. Saponifiable lipids are esters of fatty acids that can undergo saponification. This includes the triglycerides, and phospholipids. Nonsaponifiable lipids are lipids that do not contain any fatty acids or ester linkages. Examples are the steroids, prostaglandins, leukotrienes and terpenes .

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Vegetable oil is a tryglyceride obtained from plants. Castor oil is a vegetable oil extracted from the seeds of the castor oil plant ( Ricinus communis ) (“Vegetable oil” oil”, 2016). It contains ricinoleic acid, an unsaturated fatty acid and has various uses, such as increasing increasi ng hair growth, lubric lubricant, ant, and skin cleanser. This experiment aims to characterize a fat or an oil sample using the grease-spot test, saponificati saponifi cation on test, acrolei n test, and unsaturation unsaturation test. Methodology

 A. Grease-Spot Grease-Spot Test Test  A filter filter paper was obtained obtained and and four four areas were were labelled labelled as “castor “castor oil”, “lecithin”, “H₂ O”, and “dicloromethane”. “dicloromethane” . After which a drop of each sample was placed in the corresponding areas of the filter paper, with the use of Pasteur pipets. It was then subject to heat by placing it on a hot plate adjusted to its lowest setting. The The translucence of the the lipid li pid samples were observed. B. Saponification Saponificati on Test Three large-sized large-sized test tubes labelled “oil”, “fat”, and “H ₂O” were used for this test. 8 drops of each sample were placed into their corresponding test tubes. 10 drops of NaOH solution was then added to each tube, which was then placed in a boiling water bath for 15-20 minutes. After heating, it was cooled to room temperature, then 5 mL of distilled water was added to the test tubes. The tubes were stoppered using a cork and the contents were vigorously mixed, and observations were recorded. The mixtures were acidified with a few drops of H₂ SO₄  which was checked with blue litmus paper. A glass stirring rod was used to mix the contents of the tube and the material which collects on top of the solution 3

was noted. The pH of the material was recorded using a piece of blue and red litmus paper, then observations were recorded. C. Acrolein Test 1 gram of KHSO ₄  was weighed and placed in a test tube. 5 drops of the oil sample or a small piece of solid lipid was placed in the tube. The test tube was heated over a Bunsen burner for a few minutes, holding it with a test tube holder. It was allowed to cool while noting any strong odor of acrolein. D. Unsaturation Test Three large-sized large-sized test tubes labelled “oil”, “fat”, and “ glycerol” glycerol”  were used. 3 mL of dichloromethane was placed in each test tube. 10 drops of each sample were placed into their corresponding test tubes, then the contents were thoroughly mixed. 5% bromine-dichloromethane solution was added to a 50 mL buret using a funnel under the fume hood. The initial volume was recorded. Subsequently the bromine-dichloromethane solution was added drop wise from the buret to each test tube while stirring until the reddish-brown bromine color first appears. The final volume of the bromine-dichloromethane solution in the buret was recorded and the volume of the solution required for the reaction was calculated. Results and Discussion

 A. Grease-Spot Grease-Spot Test Test The grease-spot test is a general test indicating whether a sample is a lipid or not. A positive posi tive result is a visible, visib le, translucent translucent spot left by the sample. In this test, tes t, castor oil and lecithin tested positive. The dichloromethane (DCM) and H ₂O 4

samples tested negative because they do not have any lipid components. Fats are non-volatile, meaning they have high boiling points. In the test, H ₂O and DCM are evaporated. The lecithin and castor oil showed a translucent spot after placing it in the hot plate. The “translucence” observed “translucence”  observed is because light can be diffracted in the spot (Experiment 8: Grease Spot Test, 2016). Table 1 shows that all groups obtained the correct results for each sample. Table 1. Grease-spot Test. Group

Vegetable Oil

Lecithin

Dichlorom Dichloromethane

H₂O

1 Canola Oil 2 Butter 3 Sesame Sesame Oil 4 Olive Oil

Spot formed; translucent Spot remained

Spot formed; not translucent Slight spot remained

No spot; area not translucent No spot remained

No spot; area not translucent No spot remained

Spot remained

Spot remained

No spot remained

No spot remained

Spot visible, translucent grease mark Translucent grease mark Translucent grease mark

Spot less visible than olive oil

No spot visible

No spot visible

Translucent grease mark Slight translucent grease mark

 Absence  Absence of spot spot

Absence Absence of spot

No visible changes

No visible changes

Spot formed; translucent Translucent grease mark Spot formed; Translucent Translucent spot

Spot slightly seen; not translucent Slight translucent grease mark Spot formed; Slightly Translucent Translucent spot

No spot formed; evaporated No visible change

Spot formed, not translucent No visible change

No Visible spot

No Visible Spot

No translucent spot

No translucent spot

5 Corn Oil 6 Extra Virgin Olive Oil 7 Margarine 8 Coconut Oil 9 Castor Oil 10 Palm Oil

B. Saponification Saponificati on Test Saponification is the base/alkaline hydrolysis of the esters of fats or oils to afford glycerol and the salt of the corresponding fatty acid. The term literally means "soap making". The principle of this test is that fats (triglycerides), upon alkaline 5

hydrolysis (either with KOH or NaOH) will yield glycerol and potassium or sodium salts of fatty fatty acids (soap).

Figure 1. Principle of saponificati sa ponification. on.

Triacylglycerols are nonpolar, hydrophobic, insoluble and water and is bound by ester bonds (“ ( “Estimation of Saponification Value ”, 2016). 2016). In the experiment, upon the addition of NaOH, a strong base, all samples show turbid solutions. The NaOH serves to neutralize the fatty acids. After heating and shaking vigorously, the presence of bubbles and a precipitate should form (positive).  Almost  Almost all grou groups ps obtained obtained the the same observation observation in the the vegetable vegetable oils and melted melted fats. H₂ SO₄  was used to acidify and as a dehydrating agent. Upon its addition, the melted fat or oil floats on top of the solution. With the use of litmus paper, its pH is recorded and it should test positive (acidic) in the melted fat and oil and negative on the H₂ O, however, human error and mishandling of glass wares may have caused this result. Saponification test yields positives for lipids that can undergo alkaline hydrolysis, as well as those with ester bonds. Results are tabulated below:

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Table 2. Saponifi Sapo nification cation Test Te st of oil samples. Group 1 Canola Oil 2 Butter 3 Sesame Sesame Oil 4 Olive Oil 5 Corn Oil 6 Extra Virgin Olive Oil 7 Margarine 8 Coconut Oil 9 Castor Oil 10 Palm Oil

After fte r Heating He ating & Shaking Bubbles formed, cloudy solution

After fte r Acidification W hite substance substanc e on top

pH Acidic

Cloudy white with bubble formation

W hite cloudy ring at the surface

Acidic

Lesser bubble formation on the top compared to fat; cloudy solution Formation of few bubbles

Light yellow top layer; white solution W hitish material formed in the top of the whitish solution White turbid solution with yellow material Yellow Yellow top layer in a turbid solution

 Acidic  Acidic

Light yellow, thick solution

Turbid w/ light yellow layer on top

Acidic

 Appea  Appearance rance of bubbles bubbles and precipitate on top Slightly turbid, w/ bubbles

 Appea  Appearance rance of bubbles bubbles and precipitate on top W hite layer on top

 Acidic  Acidic

W hite liquid w/ bubbles

W hite layer on top

Acidic

After After Acidificati Acidification on Yellow substance on top

pH Acidic Aci dic

Yellow ring at the surface Yellow top layer; cloudy solution

Acidic Aci dic Acidic Aci dic

Yellowish material materi al formed in i n top of a white opaque solution White turbid solution with yellow ppt.

 Acidic

Yellow layer on top of turbid solution Yellow layer on top  Appearance  Appearance of bubbles bubbles and and precipitate on top Yellow layer on top Yellow layer on top

 Acidic

Upper Upper layer: yellow, lower layer: white liquid, bubble formation Presence of bubbles and yellow top layer in transparent solution.

 Acidic  Acidic  Acidic  Acidic Acidic

Acidic

Table 3. Saponification Test of Melted Fat. Group 1

2 3 4 5

6 7 8 9 10

After After Heating & Shaking Cloudy, yellowish yellowish solution. bubbles formed Cloudy yellow yellow with bubble formation formatio n Greater bubble formation formatio n at the top, suspended white particles in the sol’n. Formation Formati on of bubbles in the upper layer Upper layer: yellow yellow ppt., lower layer: yellow turbid solution, bubble formation Turbid Turbid solution with the presence prese nce of bubbles and a yellow yellow top layer Yellow-orange w/ oil oi l precipi preci pitate tate Appearance of bubbles bubbles and precipitate on top Slightly yellow turbid sol’n w/ bubbles Light Lig ht yellow yellow liquid w/ yellow yellow bubbles on top 7

 Acidic

Acidic Aci dic  Acidic Acidic Aci dic Acidic Aci dic

Table 4. Saponification Test of H ₂O. Group After After Heating & Shaking 1 Bubbles formed, clear yellowish yellowish solution 2 Clear solution w/ little bubble formation formatio n 3 Least bubble formation at upper layer layer 4 No bubbles formed 5 Transpare Transparent nt liquid 6 Transpare Transparent nt Solution 7 Light yellow yellow liquid 8 No bubbles bubbles and precipitate precipi tate on top 9 Transpare Transparent nt solution w/ bubbles 10 Clear liquid

After After Acidifi Acidification cation No formation formatio n on top Clear Clea r solution Clear solution solution Clear Solution Solution Transpare Transparent nt liquid Clear solution w/o top layer Clear solution solution No bubbles and precipitate precipi tate on top W/ slightly soluble material materi al Clear Solution Solution

pH Acidi Aci dic c Acidi Aci dic c Acidic Acid ic Acidic Acid ic Acidi Aci dic c Acidi Aci dic c Acidic Acid ic Acidic Acid ic Acidi Aci dic c Acidic Acid ic

The quantitative part of the saponification test but was not performed in the experiment involves calculating the saponification number of value of the fat. The saponification number is also called the Koettstorfer number. It is the weight (in milligrams) of potassium or sodium hydroxide required to neutralize the fatty acids resulting from the complete hydrolysis of 1 g of fat ( “Estimation of Saponification Value”, Value”, 2016). 2016). The higher saponification number, the lower the molecular weight of the lipid. Listed below are the saponification numbers of the vegetable oils used by the groups. Table 5. Saponification numbers of oil samples. Oil Sample Sample Canola Oil Butter Sesame Sesam e Oil Olive Oil Corn Oil Extra Virgin Olive Oil Margarine Coconut Oil Castor Oil Palm Oil

Saponification Number Number 173 188-195 184-196 188-193 184-196 248-265 177-185 190-205

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The two remaining tests (acrolein and unsaturation test) were not performed in the experiment, however, the principles will be discussed. C. Acrolein Test This test detects the presence of fats/glycerols. Its principle is the oxidation and dehydration reaction due to the exposure to heat (Sottrup-Jensen, 2007).  Acrolein  Acrolein is i s an unsatu unsaturat rated ed aldehy aldehyde formed formed when glycerol glycerol reacts with potassium potassium hydrogen sulfate (KHSO ₄ ). As stated previously, glycerol is the product of the hydrolysis of a lipid. The reagent KHSO ₄  then serves as a dehydrating agent. Too much heating would also cause the blackening of the mixture, and a positive result of this this test would be the the pun p ungent gent odor of the the acrolein. acro lein. D. Unsaturation Test  As the the name name suggests, suggests, th this test determin determines es the the degree of saturat saturation ion as well well as the presence of double bonds of the lipid. As mentioned in the introductions, saturated fatty acids only have single bonds while unsaturated fatty acids contain double bonds. The reagent used, bromine-dichloromethane, contains a halogen component. This halogen reacts with unsaturated fatty acids ( “Qualitative and Quantitative Tests for Lipids ”, 2016). A reddish-brown bromine color appears, indicating the presence of double bonds. Therefore, the vegetable oil, melted fat, and glycerol would test positive. The degree of saturation of the sample could be determined by the amount of bromine taken up by the lipid sample, which is why the initial and final volumes should be recorded. Furthermore, iodine can also substitute bromine in this test

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Conclusion

Saponifiable lipids were characterized in the experiment through two tests. The grease-spot test show visible positive result in the vegetable oil and lecithin. Saponification test show the formation of soaps by base hydrolysis of the esters of fats or oils to afford glycerol and the salt of the corresponding fatty acid. The presence of bubbles and precipitate and an acidic pH resulted from the vegetable oil and melted fat, indicating that it is positive. References

 Appling,  Appling, D.R., D.R., Anthon Anthonyy-Cahil Cahill, l, S.J., & Math Mathews, C.K. (2016) Biochemistry: Concepts and connections. Boston: Pearson Education Unlimited.

Experiment 8: Grease Spot Test - lungtp.com. (n.d.). Retrieved October 24, 2016, from http://www.lungtp.com/biochem/e_bcdxb.html Nelson, D., & Cox, M. M. (2005). Lehninger Principles of Biochemistry. New York: W.H. Freeman and. Qualitative and Quantitative Tests for Lipids. (n.d.). Retrieved October 24, 2016, from http://www.biologydiscussion.com/lipids/tests/qualitative-and-quantitative-testsfor-lipids/13050 Sottrup-Jensen, L. (2007). General biochemistry: Instructions for laboratory exercises.  Aarhu  Aarhus: Departmen Departmentt of Molecu Molecular lar Biology, Biology, University University of Aarhus. Aarhus. Vegetable

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http://medlibrary.org/medwiki/Vegetable_oil

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vlab.amrita.edu,. (2011). Estimation of Saponification Value of Fats/Oils. Retrieved October 24, 2016, from vlab.amrita.edu/?sub=3&brch=63&sim=688&cnt=1

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