Experiment 7: Lipid Extraction
I. Abstract
Lipids are biomolecules that are used primarily for structural components of the cell, signaling molecules and energy storage purposes. Lipids are naturally occurring esters of long chain fatty acids with both hydrophobic chains, which is insoluble to polar organic solvents and hydrophilic chains which is soluble to polar organic solvents. Because of this conformation, they can assume a wide range of complex structures including fused rings. Lipids can be isolated from cells through different techniques and their presence can be tested through different qualitative tests. The sample choice is egg yolk and was used as a source of lipids in the experiment. Liquid-liquid extraction, separation of the organic and aqueous layer was used to extract the supernatant or extract. Also, thin layer chromatography or TLC was used to separate the different lipid components by using the Rf values computed. The farther the distance traveled by the compound (higher Rf), the more nonpolar the component, while the smaller the distance traveled, the more polar the component (lower Rf). Lecithin and cholesterol was not able to travel the plate. After, the isolated lipid was subjected to qualitative tests such as Acrolein test, test for phosphates, Leibermann-Burchard test and test for unsaturation. Acrolein tests determine the presence of glycerin; the test for phosphate detects phosphate groups in the structure of the lipid, Leibermann-Burchard uses cholesterol as the standard and detects the presence of steroids and terpenes. The test for unsaturation on the other hand detects the presence of double bonds through halogenation reaction. The extract contains glycerol, phosphate, cholesterol and double bonds. Errors in the experiment can be attributed to the human error, false methodology and presence of contaminant in the reagent used.

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II. Keywords: Lipids, Thin layer chromatography, Acrolein Test, Leibermann-Burchard test, Phosphate/Unsaturation test
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III. Introduction
Lipids are hydrophobic, heterogenous biomolecules present in biological systems which is insoluble in polar organic molecules like water but soluble in organic non-polar organic solvents like methanol. (Lehninger, 2008). Lipids are naturally occurring esters of long chain fatty acids which can serve as constituents and structural components of cellular membranes that function as barriers from each other and from the environment (Campbell, 2012). They are also used as storage of energy and important signaling molecules including important vitamins and hormones, which is essential for an organism to maintain its cellular processes and functioning (Lehringer, 2008; Campbell, 2012; Stryer, 2007). Lipids include various combinations of fatty acids, ethers, terpenes, steroids, prostaglandins, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and K), monoglycerides, diglycerides, triglycerides, phospholipids and other biomolecules like glycolipids etc. (Lehringer, 2008; Campbell, 2012; Stryer, 2007). Compared to other biomolecules, lipids differ in their structures and are grouped accordingly. Some lipids can even be associated with other biomolecules. This includes lipoproteins and glycolipids. Glycolipids and lipoproteins diversify the roles of lipids in the cell. They act as biological messengers (like steroid hormones) and transport particles, respectively (Stryer, 2007).

In this experiment, the methodology was designed to determine the components of the lipids. Lipid extraction was done using the liquid-liquid procedure where compounds are separated based on their relative solubilities in the presence of two immiscible liquids (often a hydrophilic and organic solvent). Also known as partitioning, it divides the two liquid systems. It is performed using a separatory funnel (Boulder, 2013). Using this method the organic solute will dissolve in the organic solvent and the hydrophilic solute will dissolve in the hydrophilic solvent. Since the solvents are separated, the solutes will separate as well. The next procedure, which includes many qualitative tests, is done to detect the presence of lipids. This determines if the extraction was a success. The qualitative tests done on the extract include acrolein test, test for phosphate, Liebermann-Burchard test, and test for unsaturations. Acrolein test detects glycerol by the addition of KHSO4 and heat. A positive test is signaled by the formation of acrolein which is characterized by a burnt oil smell. Phosphate test is performed by incinerating and filtering the sample and adding molybdate. A positive test is signaled by a yellow precipitate. Liebermann-Burchard test detects cholesterol by the addition of acetic anhydride in the presence of sulfuric acid. A positive test is signaled by a green complex. Test for unsaturation makes use of halogenation reaction to test the degree of saturation. These tests are done to classify the lipids based on their structure and composition.

IV. Objectives The objective of the study in general is to learn the techniques and understand the principles behind isolating lipids. It specifically wants to identify the principles in using liquid-liquid extractions for isolating lipids, and by confirming the presence of lipids in the isolated samples using qualitative tests.

V. Methodology
A. Solvent extraction

The lipid-filled yolk of a chicken egg was used and combined with a 50ml, 1:2 ratio of CHCl3 and Methanol mixture. This solution was placed in a separatory funnel. The funnel contents were mixed and swirled, with occasional valve opening to release pressure accumulated inside. The funnel was left to stand until an organic layer is completely seen to be separated. The organic layer was collected while the separatory funnel was covered with cloth gauze. Acetone was added until precipitation stopped. The mixture was then centrifuged at 5,000 rpm for 10 minutes. The supernate was set aside for analysis. It was dissolved in methanol before chromatography and qualitative testing.

B. Qualitative Tests The supernate obtained from centrifugation was used as test sample. Prior to qualitative testing the test sample was dissolved in methanol.

1. Acrolein Test To 0.2 mL of test samples in a test tube, a pinch of potassium sulfate (KHSO4) was added. The resulting solution was then heated gently then vigorously. The same procedure was done to 0.2 mL of glycerol and oleic acid. They were the positive controls for this test. 2. Test for Phosphates Small amounts of the sample were incinerated in a porcelain crucible. An amount of 3mL Distilled water was added to the cooled residue. The solution was then filtered. To the filtrate, 0.5 mL (NH4)2MoO4 and 2 drops concentrated HNO3 were added. The solution was then heated and was allowed to stand. A yellow precipitate was taken note of. Lecithin was used as control for this test.

3. Liebermann-Burchard Test To 0.2mL of test sample, 0.3 mL acetic anhydride and a drop of concentrated H2SO4 was added. The solution was mixed and changes in the solution’s colour was observed and noted. Cholesterol, also dissolved in methanol, was used as control for this test.

4. Test for Unsaturation

To 0.2 mL of test sample, 1 mL CHCl3 was added. Hubl’s solution and shaking were applied to the mixture until a decolorization was observed. Number of drops added to the mixture was noted. Oleic acid was used as positive control in this test.

C. Thin Layer Chromatography

A chromatography chamber was prepared using a 1:2 methanol-acetone solvent mixture. The TLC plate was labelled and spotted with the supernate and the standards oleic acid, stearic acid, lecithin, and cholesterol. The TLC plate was left to stand inside the chromatographic chamber until the solvent was around 1cm from the top end of the plate. The plate was then taken out from the chamber and was air-dried. The spots developed by subjecting TLC plate to iodine vapors. Rf values of the different spots were then calculated using the distance of the spots from the origin.

VI. Results A. 5 4 3 2 1
Thin Layer Chromatography

Fig 1. TLC Chromatography results
Lecithin and cholesterol extraction was unsuccessful. The Rf is the ratio of the distance travelled by the substance to the distance travelled by the solvent.

Table 1. TLC results | Distance travelled | Rf | 1 Lecithin | - | - | 2 Cholesterol | - | - | 3 Extract | 2.4 cm | .3077 | 4 Stearic | 2.6 cm | .3333 | 5 Oleic | 3.1 cm | .3974 | Solvent | 7.8 cm | - | For computation of Rf values, the formula was used:
Rf= distance the sample traveleddistance traveled by the solvent

Solutions:
Extract Rf = 2.4/7.8 = .3077
Stearic Rf = 2.6/7.8 = .3333
Oleic Rf = 3.1/7.8 = .3974

B. Acrolein Test

The acrolein test detects the presence of glycerol. A positive test is characterized by a burnt grease odor.

Table 2. Acrolein results | KHSO4 + heat | Extract | Sour egg odor | Glycerol | Burnt sugar odor | Oleic acid | No change in odor | C. Phosphate Test
A positive test is characterized by the presence of a yellow precipitate.
After incinerating and filtering, (NH4)2MoO4 and concentrated HNO3 was added and was then heated.

Table 3. Phosphate results | (NH4)2MoO4 + conc. HNO3 + heat | Extract | Formation of yellow precipitate | Lecithin | Formation of white precipitate | D. Liebermann-Burchard
The Liebermann-Burchard test detects the presence of cholesterol. A positive test is characterized by the formation of a green complex.

Table 4. Liebermann-Burchard results | Acetic anhydride + conc. H2SO4 | Extract | Dark green solution | Cholesterol | Dark green solution |

E. Unsaturation
The degree of unsaturation depends on how much iodine it can absorb. The degree is measured by the number of drops needed to decolorize the solution.

Table 5. Unsaturation results | Hubl’s solution | Extract + CHCl3 | 4 drops | Oleic acid + CHCl3 | 95 drops |

VII. Discussion A. Lipid Extraction
Lipids are contained in cells, and in this experiment, the lipids are extracted from cells in an egg yolk. An average egg yolk contains different kinds of lipids. It contains cholesterol and fat-soluble vitamins like vitamin A, D, E, and K. It also contains saturated and unsaturated fatty acids like oleic acid, linoleic acid, palmitoleic acid, and linolenic acid (unsaturated) and palmitic acid, stearic acid, myristic acid (saturated). It also contains lecithins, which are fatty substances which are commonly found in animal and plant tissues. Lecithins contain phosphoric acid, choline, fatty acids, glycerol, glycolipids, triglycerides, and phospholipids. Lipids are extracted from cells of tissues using solvents. The solvent used for extraction is chosen according to the properties of the lipid to be extracted. Solubility of the lipid depends on the structure of their polar and/or nonpolar moieties. This experiment used an extraction solution consisting of chloroform and methanol. 1. CHCl3 & MeOH – The solution consisting chloroform and methanol is considered one of the most general solvents used for lipid extraction.

B. Thin Layer Chromatography
Samples from the extract are subjected to chromatography using the solvent (chloroform and methanol) against a chromatography paper along with other known examples. The mobile phase used is the solvent while the stationary phase is paper. The extract will have many different lipids. Different lipids will have different structures and therefore, different retention factors. The extract was run with standards (pure, known samples of lipids) to determine if such standards are present in the extract.

C. Qualitative Tests
The lipids extracted are needed to be identified, also, it is essential to know if a certain extraction solvent or technique is useful. Since lipids have different structures, different tests are used for different kinds of lipids and for identifying structural components of lipids.

1. Acrolein Test – Triacyl glycerols contain fatty acids and glycerol. In acrolein test, glycerol is dehydrated by heating and the addition of KHSO4 and acrolein is produced. Acrolein has the characteristic burnt oil odor that characterizes a positive result for the test.

Fig 2.1 Acrolein Test Reaction

2. Test for Phosphates – Some lipids contain phosphates (phospholipids). Phosphates can be tested for by adding molybdate ions to the solution to form yellow precipitates, ammonium phosphomolybdate.

12MoO42- + 3NH4+ + PO43- + 24H+ → (NH4)3[P(Mo12O40)] + 12H2O
Fig 2.2 Ammonium Phosphomolybdate reaction

3. Liebermann -Burchard – The extract is reacted with acetic anhydride and sulfuric acid to produce a solution with transitioning colors which finalizes as a dark green solution. The hydroxyl group is removed from the A ring of the cholesterol which causes different degrees of conjugations, hence the different color transitions. The color finalizes as dark green as the molecules stabilizes with a hybrid structure.

Fig 2.3 Liebermann-Burchard Reaction

4. Test for Unsaturation – The test for unsaturation follows a simple halogenation reaction for an alkene. A nucleophilic attack on the double bond by the halogen creates a bridged intermediate until it is attacked again by the halide and forms a halogenated product.

Fig 2.4 Test for Unsaturation Reaction

VIII. Conclusions and Recommendations

Lipids are biomolecules which do not have a structural monomer but instead have a basic formation of a polar head and non polar carbon chain that can have double bonds. Extraction of lipids can be done by liquid-liquid extraction through getting the supernate and not the precipitate. Thin layer chromatography was used to identify the properties of the lipids in the experiment. Non-polar components travel faster compared to polar components as the non-polar are more attracted to the polar plate. Lecithin and cholesterol was unable to travel in the plate. Oleic acid has the highest Rf value, thus, show same polarity.

Based on the qualitative tests such as the Acrolein test, Test for Phosphates, Leibermann-Burchard Test and Test for Unsaturation the extract contains glycerol, phosphate, cholesterol and double bonds, respectively.

Errors and false results in this experiment can be attributed to human errors and experimental errors like inefficiency of lipid extraction because of using wrong reagents, contamination of reagents or malpractice of experiment methodology and the mistake of the methodology itself. However, it can be implied that egg yolk is an efficient source of lipids since it is rich in the compounds most important for life like fats, carbohydrates and cholesterol; lecithin, basis of the entire test performed in the sample lipids.

IX. Guide Questions
1. What is the function of the components of the solvent used for extracting lipids in biological samples? The solvent components include methanol, chloroform and acetone. The methanol-chloroform mixture was used to solubilize the desired lipids into one layer which can be separated using a separatory funnel. Chloroform interacts with water by forming weak hydrogen bonds and methanol solubilizes the lipids’ polar components that are supposed to be separated. This method separates the mixture into two layers: an aqueous and organic layer (the latter containing the lipids). Acetone helps dissolve other simple lipids present in the sample.

2. If you were extracting lipids from myelin sheaths, will you use the same CHCl3: MeOH ratio of 1:2? Why or why not? Yes. This is because the lipids in myelin sheaths which are sphingomyelin lipids are also phospholipids. This means their characteristics are similar to those in the sample.

3. What reaction with I2 vapor results in the appearance of lipid spots in chromatography? Iodine reacts with the double bonds present in lipids. After which, a complex is formed which makes the color spots seen in the chromatography paper.

4. Why should the sample used for the test for phosphates be incinerated? This is because burning will destroy the organic parts of a phospholipid structure and free the phosphate component. The latter can then react with the ammonium molybdate reagent and can cause a yellow precipitate that confirms their presence.

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