Lab-6 (1)

Ateneo de Zamboanga University College of Science and Information Technology Natural Sciences Department NURBIO D ______________________________________________________________________________ Name/s: AREVALO, Alessandra L. BERNARDO, Alexander LUI, Sharah Gayle P. PASCUA, Precious SALIBAY, Chosen Lloyd Group No. 6 Section: BSN - D

Scores Introduction _______ Objective/s _______ Results and Discussion _______ Questions _______ Conclusion and Insights _______ Total _______

Activity No. 6 Lipids I.

Introduction

Lipids are found in living organisms. They are a family of substances that are insoluble in water, which means they are generally hydrophobic. Lipids play three major roles in human biochemistry. They store energy within fat cells, they are parts of the membrane that separate compartments of aqueous solution from each other and they serve as chemical messengers. Animal fats and plant oils are triglycerides. Triaglycerides are the form of storage of fatty acids in organisms, they are triesters of fatty acids and glycerol. Those triaglycerides that are solid are called fats, while those that are liquid are called oils. Phospholipids are lipids that contain phosphate groups. Lipids are major components of biological elements, and the major classes include the glycerophospholipids, sphingolipids and glycospingolipids in animals. Glycerolipids are widespread in plant and bacterial membranes. Lipids can be classified as saponifable and nonsaponifable. Saponifable lipids are esters of fatty acids which includes triglycerides and phospholipids, while nonsaponifable lipids are lipids that do not contain any fatty acids or ester linkages which includes, steroids, prostaglandins, leukotrienes, and terpenes. II.

Objectives 1. To compare the different tests used in determining lipids. 2. To distinguish the different properties of lipids through the different test performed and different reagents used. 3. To detect the presence of lipids through the different tests

III.

Results and Discussion

A. Solubility Tests Sample

Cottonseed Oil

Solvent Used

Distilled water (A)

Result

Immiscible, polar Time: Greater than 5 minutes Negative Result

Ethyl alcohol (B)

Partially immisicible, polar Time: Greater than 4 minutes Negative Result

Ether (C)

Immiscible, slightly polar Time: 10 seconds Negative Result

Chloroform (D)

Miscible, non-polar Time: 2 minutes Positive Result

5% HCl (D)

Immiscible, polar Time: Greater than 3 minutes

Negative Result

5% NaOH (E)

Miscible, non-polar Time: 3 seconds Positive Result

In our experiment for the Solubility Test, we first followed the procedure in performing this experiment, which was to pipette 1mL of the following solvents in separate test tubes: distilled water, ethul alcohol, ether, chloroform, 5% hydrochloric acid, and 5% sodium hydroxide. From a pipette, we added 1-2 drops of cottonseed oil in each test tube and shake thoroughly. We then recorded the time required for the oil to dissolve.

In the experiment, we observed that Distilled Water (A), Ethyl Alcohol (B), Ether (C) and 5% HCL (E) became immiscible, which is a polar solvent, resulting to a negative result. While the

Chloroform (D), and 5% NaOH (F) became miscible, which is a non-polar solvent, resulting to a positive result in the Solubility Test.

In the Solubility Test, it is the preliminary test which detects the presence of all lipids. This test detects the solubility of lipids in various solvents to check wherether it is miscible or immiscible in polar or non-polar solvents. It is based on the property of lipid to dissolve in differet solvents. Lipids are readily miscible in non-polar solvents like chloroform, partially soluble in a polar system like ethanol and immiscible in a polar solvent like water. For a positive result, lipids are soluble in non-polar solvents. A non-polar solvent contains bond atoms with similar electronegatives. While for a negative result, lipids are insoluble in a polar solvent. A polar solvent have large dipole movements, they contain bonds between atoms with different electronegatives.

B. Test for Unsaturation Name of Test

Test for Unsaturation

Test Solutions

Reagents Added

Result

Oleic Acid

Br2 in CCl4

It took 3 drops to produce a faint color of yellow, at around 8 drops the color yellow became very noticeable at this point. There was no presence of orange color.

(D)

Palmitic Acid

CHCl3 Iodine

Haus It took 30 seconds for the color to spread.

CHCl3 Iodine

Haus It took 40 seconds for the color to spread.

CHCl3 Iodine

Haus It took 40 seconds of the color to spread.

(A)

Oleic Acid (B)

Cottonseed Oil (C)

In our experiment for the Tests for Unsaturation of Fatty Acids, we first followed the procedure in performing this experiment, to 6 drops of carbon tetrachloride, add 3 drops of oleic acid. Then add bromine water in carbon tetrachloride drop by drop into the mixture, shaking vial after each addition. Then, into each of 3 test tubes, we placed 2 mL of chloroform. Next we added to each test tube 0.2 g of palmitic acid, 4 drops of oleic acid, and 4 drops of cottonseed oil respectively. We shaked each test tube thorougly, then we added 4 drops of Hanus – iodine solution

to each test tube. Afterwards, we recorded all results, and time in seconds for the color to disappear.

In the experiment, for the Oleic Acid, it took around 3 drops of Br2 in CCl4 to have a faint color of yellow, and then on the 8th drop, the color yellow in the solution became more noticeable. For the Palmitic Acid, it took around 30 seconds for the color to spread. Then for the Oleic Acid, it took around 40 seconds for the color to spread. Lastly, for the Cottonseed oil, it took around 40 seconds for the color to spread. All solutions produced a color of pink which resulted to a positive result.

In the Test for Unsaturation, it is used to detect the presence of unsaturated fatty acids or the amount of double bond in a liquid sample. All the neutral fat contains glycerides or fatty acids. The double bond found in the structure of unsaturated fatty acids which becomes saturated by either taking up bromine or iodine. If the lipids are more unsaturated fatty acids or more double bonds, that means it will take more iodine. For a positive result, the outcome will be a pink color that will disappear by the addition of unsaturated fatty acids. While for the negative result, the outcome is that the pink color will not disappear.

C. Acrolein Test Name of Test

Acrolein Test

Test Solutions Glycerol

Reagents Added

Result

KHSO4 (s)

The solution turned into brown-red after being heated into a low flame.

(A)

A strong produced.

pungent

smell

was

Positive Result

Cottonseed oil (B)

KHSO4 (s)

The solution became transparent in color after being heated into a low flame. No strong produced.

pungent

smell

was

Negative Result

In our experiment for the acrolein test, we first followed the procedure, which was to prepare two test tubes. For the first test tube, it was to place 2 drops of glycerol and a pinch of KHSO4, while for the second test tube, place 2 drops of cottonseed oil and a pinch of KHSO4. We heated each test tube over a low flame, and then we recorded the order that was produced from the solutions.

In the results that we recorded from the experiment, we noticed that in the first test tube, after heating it resulted to a color of brown-red and it produced a pungent smell. While for the second test tube, the color became transparent, and there was no pungent smell that was produced. Between the two test tubes, the first test tube which contains glycerol and potassium bisulfated resulted to positive, wherein there is a presence of glycerol and fats, since it produced a pungent smell compared to the second test tube. In the Acrolein Test, it is used to detect the presence of glycerol and fats. This test is based on the “Dehydration Reaction”, where the water molecules removed from the glycerol by the addition of reagent potassium hydrogen sulphate. The reaction between glycerol and potassium hydrogen sulphate results in the formation of “Acrolein” that is characterized physically by the release of the pungent smell.

D. Rancid Test Name of Test Rancidity Test

Test Solutions

Reagents Added

Fresh coconut oil

Phenolphthalein The color became light pink, a bit of purple. (A)

Methyl orange

Result

The color became light orange.

(B)

pH paper (C)

Rancid coconut oil

The litmus transparent.

paper

became

Phenolphthalein The color became dull yellow, a bit of brown. (D)

Methyl orange (E)

pH paper (F)

The color resulted to a golden yellow.

The blue pH paper reacted violet, the red remained

In our experiment for the Rancidity Test, we first followed the procedure in performing this experiment, which was to prepare six test tubes, three test tubes for the coconut oil, and three test tubes for for the rancid oil. On the first test tube, we placed 5 drops of coconut oil, and 5 drops of rancid oil. Next, we tested the reaction of fresh coconut oil with 1-2 drops phenolphthalein, 12 drops methyl orange, and red and blue litmus paper. Then, we did the same reaction test with rancid

oil.

Afterwards,

we

recorded

the

results

from

the

tests.

In the first test with fresh coconut oil, when mixed with the reagents, there was no oxidation in the outcome. For the phenolpthalein, it became pink which means that the oil was basic. Then for the methyl orange, it resulted with a color of light yellow, which resulted that the coconut oil was still basic. Lastly, for the litmus paper, both resulted to becoming transparent. While for the second test with the rancid oil, the pheolphthalein resulted light yellow which signfies as pH 6-7 which is near to basic. Then for the methyl orange, it turned golden yellow which means the solution increased in acidity. And lastly, for the litmus paper, the blue litmus paper turned purple as a result of it being acidic, while the red litmus paper remained as it is. Rancidity is a term generally used to denote a condition of unpleasant odors and flavours in food resulting from the deterioration in the fat or oil portion of a food. In the Rancidity Test, in knowing whether an oil is rancid, it is by the development of unpleasant smells in fats and oils that

are

often

accompanied

by

the

changes

in

their

texture

and

appearance.

E. Saponification

Name of Test

Test Solutions

Saponification coconut oil

Reagents Added

Result

10% KOH

The whole procedure took 25 minutes. The result of the coconut oil became clear, an oil at the top.

In doing our experiment for the Saponification test, we first followed the procedure, which was to weigh 1.5 g of fresh coconut oil in a dry beaker, then we added 10 mL of 10% alcoholic potassium hydroxide. Then, we covered the beaker with a watch glass. Afterwards, we fill yje watch glass with crush ice, boil it over a water bath until a drop of the hot solution aaded to cold water in a test tube does not form globules of fat. Then, we removed the watch glass to drive off the alcohol, next we added 20 mL of water to the gelatinous mass and warm the solution to dissolve the

soap.

Afterwards,

we

observed

and

recorded

the

results.

In doing the Saphonification test, we followed the procedure that took around 25 minutes in order to produce the outcome of a froth that appeared in the test tube signifying that it was a positive result. In the Saponification test, it is a test based on the “Saponification reaction”, where the triglycerides of lipid react with an alkali NaOH (Sodium Chloride) and produce soap and glycerol in the presence of ethanol. This reaction also refers to as “Alkaline hydrolysis of ethers”. For a positive result, a froth must appear in the test tube. While for the negative result, there is an absence of froth in the test tube.

F. Properties of Soap Name of Test Salting Out

Test Solutions Soap Solution (A)

Formation of Fatty Acids

Soap Solution

Reagents Added

Result The NaOH (Salt) diffused on the test tube when mixed with the solution from the previous experiment. Some floated at the top of the test tube. There was a formation of crystals; white substance The solution became cloudy, and there was a formation of bubbles in the test tube

(B)

Insoluble Soaps

Soap solution

5% Calcium There was a formation of white suds Chloride that looked cloudy

(C) Soap Solution

5% Magnesium There was a formation of white suds Chloride that looked cloudy

(D) Detergent (E) Detergent (F)

5% Calcium There was a formation of white suds Chloride that looked cloudy

5% Magnesium It produced white precipitation, suds Chloride were formed

In doing our experiment for the Properties of Soap, we first followed the procedure for Salting Out, Formation of Fatty Acids, and Insoluble Soaps. For the Salting out, we placed 10 mL of the soap solution in a beaker and added table salt gradually with stirring until no more table salt dissolves. Then we removed the solid soap formed from the liquid and washed it with water. We transferred a small amount of solid soap into a test tube and shaked it with distilled water to form suds. Second, for the Formation of Fatty Acids, we first placed 5 mL of the soap solution in

a test tube and added 10% HCl until a precipitate forms. Lastly, for Insoluble soaps, we placed 2 test tubes, each containing 4 mL of distilled water and 1 mL soap solution. To test tube 1, we added 5 drops CaCl2 solution. To test tube 2, we added 5 drops 5% MgCl2 solution. We observed the results and repeated the test using liquid detergent instead of soap solution. We observed and recorded the results, afterwards, we compared it with the results of the first part. For the Salting Out, it is used to investigate the effect of a NaCl on soap solubility. It is to get the soap out of the solution by salting out, when added sodium chloride (NaCl – Salt) to the solution until saturation; separated soap in the form of insoluble and floats above the surface. The NaCl solution provides Na+ and Cl- ions that bond to the polar water molecules and help separate the water from the soap. This process is called salting out the soap. For Insoluble soaps, it is used to investigate the effect of different cations on soap solubility. Working calcium, magnesium, lead or iron ions to the deposition of soap and make it insoluble in water, where some of these ions replace the sodium or potassium ions are present in the soap. Due to the hard water, it contain significant quantities of Ca2+, Mg2+, and some Fe3+ that react with the charged ends of the soaps to form insoluble salts of fatty acids. The insoluble salts of fatty acids Mg2+ and that Ca2+ and Mg2+ form with soap anions cause white precipitate to form.

According to Wikipedia, due to their opposite polarity, water by itself cannot penetrate grease or oil. However, when grease or oil (non-polar hydrocarbons) are mixed with a soap-water solution, the soap molecules work as a "bridge" between polar water molecules and non-polar oil molecules. Soap molecules are amphipathic and thus have both properties of non-polar and polar at opposite ends of the molecule. The oil is a pure hydrocarbon so it is non-polar. The non-polar hydrocarbon tail of the soap dissolves into the oil. That leaves the polar carboxylate ion of the soap molecules are sticking out of the oil droplets, the surface of each oil droplet is negatively charged. As a result, the oil droplets repel each other and remain suspended in solution (this is called an emulsion) to be washed away by a stream of water. The outside of the droplet is also coated with a layer of water molecules. This is also similar to a micelle which works with the same principlesthe

center

of

the

micelle

would

contain

the

oil.

F. Emulsifying Action of Lecithin Name of Test Emulsifying Action

Test Solutions 2% albumin

Reagents Added Cholesterol (A)

Cholesterol – lecithin (B)

Result Turned white in color. No odor was produced after the two solutions were mixed. Layers were formed; soluble in albumin. Negative Result Produced light brown in color. No strong odor was produced There was no layer formed, and there was a mixture between the cholesterol and albumin. Positive Result

In our experiment for the test of Emulsifying Action of Lecithin, we first followed the procedure in performing this experiment, in each of two test tubes, then we placed 5 drops of 2% albumin solution. To the test tube 1 – we added a pinch of cholesterol and shaked the tube. While to test tube 2 – we added a pinch of cholesterol and five drops of lecithin. We then compared and recorded the results.

In our experiment for the Emulsifying Action of Lecithin, in the first experiment for the 2% albumin and cholesterol, layers were formed, it was soluble in albumin. While for the second experiment for the 2% albumin and cholesterol – lecithin, there was no layer formed and it resulted to a mixture for the cholesterol – lecithin and albumin. In the first experiment, it resulted to a negative

result,

while

for

the

second

experiment,

it

was

a

positive

result.

In the Emulsification Test, it is used to detect the presence of lipids. Emulsification is the process which stabilizes the water and oil emulsion, by the help of emulsifying agents. The lipid or oil in water appears on the top of the water because of the high surface tension of water which gets together to form a separate layer. The emulsifying agents emulsify the lipid by which the lipid appears as the tiny droplets suspended in the solution. For a positive result, it gives a permanent or stable emulsion of lipid and water. While for the negative result, the oil in water emulsion will form at the top, due to the high surface tension of water.

Questions 1. Why are fatty acids insoluble in water? Fatty acids are insoluble in water since there are more hydrocarbons which are more hydrophobic than the carboxyl group which is soluble in water. The longer the chain of the fatty acid becomes, the more soluble it gets.

2. Explain why the cis-form is the predominant configuration of unsaturated fatty acids? Cis-form configuration is more predominant in unsaturated fatty acid since most of the fatty acids are in liquid form, and most of these liquid form unsaturated fatty acids has this configuration. 3. What type of rancidity occurs in vegetable shortenings? How can it be prevented? The type of rancidity that occurs in vegetable shortenings is the oxidation rancidity. In this ‘oxidation rancidity’, the oxygen molecules interact with molecules of the oil which causes damage or changes to the substance. The easiest

way to prevent this from happening is to to store these vegetable shortenings in a dark, cool place where it is less exposed to oxygen.

4. Show the structure of the parent compound of cholesterol.

5. Explain

the

cooperative

effect

of

lecithin

and

albumin.

Lecithin and albumin are good emulsifiers because they both have a nonpolar and a polar portion which helps reduce the immiscibility of two substances. When they work hand-in-hand, they are able to hold polar substances through their hydrophilic molecules, thus making the immiscibility of the two substances positive. IV.

Conclusion and Insights Lipids are fats that are important part of cells and sources of energy. Lipids are fat-like

substances for they contain a large heterogenous group of unrelated physiological and chemical substances classified together which also observes the rule “like dissolves like”. The polar end of the fatty acid is carboxyl group (R-COOH) which helps with the polarity of the lipids. The hydrocarbon chains are the reason why lipids are nonpolar meaning it makes them insoluble in polar solvents like water. The different test performed in the lipids determined some of its properties. The first test is the solubility test, the test for presence of lipid and if it is miscible and immiscible in polar or

nonpolar solvents. It showed that distilled water, Ethyl alcohol, Ether and 5% HCL became immiscible, which is a polar solvent, resulting to a negative result. Thus, the dissolving process of different solvents is based on the property of lipid. The test for unsaturation of fatty acids determined the varying number of double bonds that each fatty acid (lipid) contain. It showed that the Oleic acid took 8th drop for the color yellow in the solution to be more visible or noticeable. The Palmitic acid, it took only 30 seconds to spread; however, 40 seconds it took for the color to spread in Oleic Acid and Cottonseed oil. Thus, all the solutions resulted to positive because it produces a color of pink. In the acrolein test, the test tube 1 (2 drops of glycerol and a pinch of KHSO4), a color of brown-red and with a pungent smell that indicates a positive result. Rancidity test is the test to determine the rancidity of the oil is. It showed that phenolphthalein became pink that indicates the oil was basic. The methyl orange showed color of light yellow that indicates oil was still basic. Lastly, it showed that both became transparent. For the second test, phenolphthalein resulted light yellow and indicates near to basic and for the methyl orange it resulted to golden yellow that indicates that solution has increase in acidity. Saponification is where the triglycerides of lipid react with an alkali NaOH (Sodium Chloride) and produce soap and glycerol in the presence of ethanol. It resulted that the procedure that took around 25 minutes in order to produce the outcome of a froth that appeared in the test tube signifying that it was a positive result. Salting out is a purification method that relies on the solubility of the protein. It is to get the soap out of the solution by salting out, when added sodium chloride (NaCl – Salt) to the solution until saturation. For Insoluble soaps, it is used to investigate the effect of different cations on soap solubility. Lastly, the emulsification test is use to detect the presence of lipids. The first experiment showed negative result, while the second experiment showed a positive result because there was no layer formed on top.

V.

Referrences

Lab Activity 5 Lipids. (n.d.). Retrieved July 24, 2019, from https://studylib.net/doc/9790812/labactivity-5-lipids Lipids Chemlab Report. (n.d.). Retrieved July 24, 2019, from https://www.vbook.pub.com/doc/90427181/Lipids-Chemlab-Report Memije-Cruz, L. (2018, April 25). Qualitative and quantitative tests for lipids. Retrieved July 24, 2019, from https://www.slideshare.net/memijecruz/qualitative-and-quantitative-tests-for-lipids N, S. (2019, May 28). What is Qualitative Analysis of Lipids? definition & methods. Retrieved July 24, 2019, from https://biologyreader.com/qualitative-analysis-of-lipids.html#DichromateTest Qualitative and Quantitative Tests for Lipids. (2015, October 16). Retrieved July 24, 2019, from http://www.biologydiscussion.com/lipids/tests/qualitative-and-quantitative-tests-for-lipids/13050 Wikibooks. (2019). Structural Biochemistry/Lipids/Soap. Retrieved July 24, 2019 from https://en.wikibooks.org/wiki/Structural_Biochemistry/Lipids/Soap