Extraction of Total Lipids from Chicken Egg Yolk, Column Chromatography and Qualitative Tests for Lipids

EXTRACTION OF TOTAL LIPIDS FROM CHICKEN EGG YOLK, COLUMN CHROMATOGRAPHY AND QUALITATIVE TESTS FOR LIPIDS Maria Feliza C. Abesamis, Marie Em Clarisse P. Acosta, Francheska M. Agustin, Mary Christelle G. Aquitania and Marilu Jane H. Bagsican Group 1 2E Medical Technology Biochemistry Laboratory

ABSTRACT The experiment was about extraction of total lipids from chicken egg yolk, column chromatography of lipids and qualitative tests for lipids. Total lipids were extracted from the chicken egg yolk using 1M NaCl, isopropyl alcohol and petroleum ether. The mixture was left standing for five minutes. The lower layer was collected and subjected into column chromatography. The column used for column chromatography was packed with slurry of 0.5 g silica gel in 4 ml of petroleum ether with tapered end glass wool. For this experiment, the column was washed three times using different eluents. The first eluent used was of 9:1 mixture of petroleum ether and ethyl ether, the second eluent was 5 ml 5% methanol in dichloromethane and the last eluent was 5ml CH 2Cl2: CH3OH: H2O (1:3:1). Eluates for each eluent introduced into the column were collected in separate test tubes. Collected eluates were subjected for the qualitative tests for lipids. 10 drops of eluates were subjected for each qualitative test. The qualitative tests performed were as follows: test for ester, test for glycerol also known as acrolein test, test for phosphate, test for cholesterol also known as Liebermann-Burchard test, test for Test for α- amino acids (Ninhydrin Test) and test for lipid unsaturation with bromine. Test for esters yielded a yellow solution for the first and second eluate and a burgundy solution for the third eluate. Test for glycerol produced no odor for the three eluates. Test for phosphate produced a turbid solution for the first eluate, formed white/ slightly yellowish crystals for the second eluate and turbid yellow with crystals for the third eluate. Test for cholesterol or Liebermann-Burchard test did not show any color change for the first and third eluate while it produced a green color on the second eluate. Test for Ninhydrin formed a red orange solution for the first eluate and no color change in the second and third eluates. Triglyceride or triacylglycerol, cholesterol and phospholipid (Lecithin) were the first second and third eluates identified respectively. In the test for lipid unsaturation with bromine, 10 drops in the first, second and third eluates, 22 drops in coconul oil, 71 drops in canola oil, 90 drops in corn oil, 74 drops in olive oil and 8 drops in vegetable oil of 5% of Br 2 in CH2Cl were added before reddish brown color persisted.

INTRODUCTION Lipids are substances found in living organisms that are insoluble in water but soluble in non polar solvents and solvents of low polarity. This lack of solubility in water is an important property because our body chemistry is so firmly based on water. Most body constituents including carbohydrates which are soluble in water. But the body also needs insoluble compounds for many purposes, including the separation of compartments containing aqueous solutions from each other, that’s where lipids come in. The water-insolubility of lipids is due to the fact that the polar groups they contain are much smaller than their alkane-like (nonpolar) portions. These nonpolar portions provide the water-repellent, or hydrophobic, property. An important use for lipids, especially in animals, is storage of energy. Plant store energy in form of starch. Animals including

humans find it more economical to use lipids (fats) instead. Although our bodies do store some carbohydrates in the form of glycogen for quick energy when we need it, energy stored in the form of fats is much more important. The reason is simply that the burning of fats produces more than twice as much energy as burning of an equal weight of carbohydrates.

Composition of Chicken Egg Yolk The yolk makes up about 33% of the liquid weight of the egg; it contains approximately 60 calories, three times the caloric content of the egg white. One large egg (50 grams in weight, 17 gram yolk) contains approximately: 2.7g protein, 210 mg cholesterol, 0.61g carbohydrates and 4.51g total fat. (USDA National Nutrient Database) All of the fat soluble vitamins, (A, D, E and K) are found in the egg yolk. Egg yolks are

one of the few foods naturally containing vitamin D. The composition (by weight) of the most prevalent fatty acids in egg yolk is typically as follows: Unsaturated fatty acids: • • • •

Oleic acid 47 % Linoleic acid 16 % Palmitoleic acid 5 % Linolenic acid 2 %

Saturated fatty acids: • • •

Palmitic acid 23 % Stearic acid 4 % Myristic acid 1 %

the adsorbent. Just as in TLC, there is an equilibrium established between the solute adsorbed on the silica gel or alumina and the eluting solvent flowing down through the column.

Stationary phase (adsorbent) The stationary phase or adsorbent in column chromatography is a solid. The most common stationary phase for column chromatography is silica gel, followed by alumina. Cellulose powder has often been used in the past. Also possible are ion exchange chromatography,reversedphase chromatography (RP), affinity chromatography or expanded bed adsorption (EBA). The stationary phases are usually finely ground powders or gels and/or are microporous for an increased surface, though in EBA a fluidized bed is used.

Egg yolk is a source of lecithin, an emulsifier and surfactant. The yellow color is caused by lutein and zeaxanthin, which are yellow or orange carotenoids known as xanthophylls.

Figure 1. Chicken egg yolk Column Chromatography Column chromatography is one of the most useful methods for the separation and purification of both solids and liquids when carrying out small-scale experiments. Column chromatography is another solid-liquid technique in which the two phases are a solid (stationary phase) and a liquid (moving phase). The theory of column chromatography is analogous to that of thin-layer chromatography. The most common adsorbents - silica gel and alumina - are the same ones used in TLC. The sample is dissolved in a small quantity of solvent (the eluent) and applied to the top of the column. The eluent, instead of rising by capillary action up a thin layer, flows down through the column filled with

Figure 2. Column Chromatography Mobile phase (eluent) The mobile phase or eluent is either a pure solvent or a mixture of different solvents. It is chosen so that the retention factor value of the compound of interest is roughly around 0.2 - 0.3 in order to minimize the time and the amount of eluent to run the chromatography. The eluent has also been chosen so that the different compounds can be separated effectively. The eluent is optimized in small scale pretests, often using thin layer chromatography (TLC) with the same stationary phase.

A faster flow rate of the eluent minimizes the time required to run a column and thereby minimizes diffusion, resulting in a better separation, see Van Deemter's equation. A simple laboratory column runs by gravity flow. The flow rate of such a column can be increased by extending the fresh eluent filled column above the top of the stationary phase or decreased by the tap controls. Better flow rates can be achieved by using a pump or by using compressed gas (e.g. air, nitrogen, or argon) to push the solvent through the column (flash column chromatography). The particle size of the stationary phase is generally finer in flash column chromatography than in gravity column chromatography. For example, one of the most widely used silica gel grades in the former technique is mesh 230 – 400 (40 – 63 µm), while the latter technique typically requires mesh 70 – 230 (63 – 200 µm) silica gel. A spreadsheet that assists in the successful development of flash columns has been developed. The spreadsheet estimates the retention volume and band volume of analytes, the fraction numbers expected to contain each analyte, and the resolution between adjacent peaks. This information allows users to select optimal parameters for preparative-scale separations before the flash column itself is attempted. The objectives of the experiment are as follows: (1) to extract total lipids from chicken egg yolk, (2) to analyze the lipids present in the crude extract using column chromatography (3) to identify lipids present in each of the fractions using qualitative tests and, (4) to determine the degree of unsaturation of lipids by bromine test.

2. Extraction of total chicken egg yolk

lipids

from

The following were used for the Extraction of total lipids from chicken egg yolk: Chicken egg yolk, 5 volumes of 1M NaCl, 3ml isopropyl alcohol and 2ml petroleum ether.

3. Column Chromatography of Lipids

The following were used for the column chromatography of lipids: extracted total lipids from chicken egg yolk, 0.5 g silica gel, 4ml of petroleum ether, 5ml of 9:1 mixture of petroleum ether and ethyl ether, 5 ml 5% methanol in dichloromethane, and 5ml CH2Cl2: CH3OH: H2O (1:3:1).

4. Test for Ester

The following were used for the test for ester: EtOH: 0.5 ml of 1-BuOH (3:1) with 10 drops of eluate, EtOH: 0.5 ml of 1-BuOH (3:1), 2 drops each of 2M hydroxylamine hydrochloride and 3M NaOH, 2 drops of 6M HCl, 1 drop of 5% FeCl3. 6 H2O in 0.1M HCl.

5. Test for Glycerol (Acrolein Test)

EXPERIMENTAL A. COMPOUNDS TESTED (SAMPLES USED)

The following were used for the test for glycerol (Acrolein Test): 10 drops of eluate and pinch amount of KHSO4.

1. Samples to be tested: The following samples were subjected to test using column chromatography and qualitative test for lipids: Chicken egg yolk, Coconut oil, Canola oil, Corn oil, Olive Oil, Vegetable oil, Cholesterol and Lecithin.

6. Test for Phosphate

The following were used for the test for phosphate: 10 drops of eluate, 0.5 ml of 10% Mg(NO3)2, 6H2O in 95% EtOH,

Mg(NO3)2, 0.5 ml of 2M HCL, 0.5ml of 6% (NH4)2MoO4 and 4M HNO3.

7. Test for Cholesterol Burchard Test)

(Lieberman-

The following were used for the test for cholesterol (Lieberman-Burchard Test): 10 drops of eluate, 0.25 ml CHCl3, 6 drops of acetic anhydride and 2 drops of concentrated H2SO4.

8. Test for α- amino acids (Ninhydrin Test)

The following were used for the test αamino acids (Ninhydrin Test): 10 drops of eluate and 5-8 drops of ninhydrin reagent.

9. Test for Lipid Unsaturation with Bromine

The following were used for the lipid unsaturation test with bromine: 10 drops of eluate, 3ml CH2Cl2, 5% Br2 in CH2Cl2, 8 drops each of coconut, canola, corn and olive oil.

B.

PROCEDURES:

1. Extraction of Total Lipids from Chicken Egg Yolk The egg yolk was separated from the chicken egg and its volume was determined. It was then diluted with 5 volumes of 1M NaCl. After dilution, 2ml of the diluted egg yolk was mixed with 3ml isopropyl alcohol in a separate clean test tube. Petroleum ether with a volume of 2 ml was then added. It was covered with rubber stopper and was ensured to be well mixed. The mixture was then allowed to stand for 5

minutes until two layers were formed. After 5 minutes, the lower layer was collected and was transferred to another clean test tube. Two-dimensional thin layer chromatography (TLC) and column chromatography (CC) was performed using the lower layer.

2. Column Chromatography of Lipids Small column was prepared by pouring a slurry of 0.5 g silica gel in 4ml of petroleum ether into the glass column (Pasteur pipette) with a tapered end plugged with glass wool. The lipid extract from chicken egg yolk with a volume of 1 ml was then introduced into the column, saving the run-through in a clean test tube. The column was washed with 5ml 9:1 mixture of petroleum ether and ethyl ether, collecting the eluate in the same tube as the runthrough. The column was again washed with the second eluent (5 ml 5% methanol in dichloromethane) the eluate was then collected in another clean test tube. The column was washed with the last eluent, 5ml CH2Cl2: CH3OH: H2O (1:3:1) and eluate was collected in another test tube. The different eluates culled were saved for qualitative analysis.

3. Test for Ester

EtOH: 1-BuOH (3:1) with a volume of 0.5 ml was introduced into the 10 drops of eluate. 2 drops each of 2M hydroxylamine hydrochloride and 3M NaOH was sequentially added and was mixed well. The samples were allowed to stand for 5 minutes. 2 drops of 6M HCl was added with 1 drop of 5% FeCl3. 6 H2O in 0.1M HCl and was ensured to be well-mixed. Color was noted. Samples with positive results gave a burgundy color.

4. Test for Glycerol (Acrolein Test) A pinch amount of KHSO4 was added to 10 drops of the eluate in the test tube. Test tube was heated in a boiling water bath and odor produced was noted. Burned fat odor was observed for positive test results.

5. Test for Phosphate A volume of 0.5 ml of 10% Mg(NO3)2 with 6H2O in 95% EtOH was added with 10 drops of eluate. The test tube was placed in a boiling water bath until the solvent was evaporated and the Mg(NO3)2 was decomposed. The test tube was removed from the bath when the white residue was formed and brown gas stopped evolving. 2M HCL with a volume of 0.5ml was added and was mixed to dissolve the solid residue. Test tube was then heated in a boiling water bath for 5 minutes. 0.5ml of 6% (NH4)2MoO4 was added with 4M HNO3. Color change was noted. Positive results for this test were formation of a yellow color followed by formation of a fine yellow precipitate. This indicated the presence of phosphate.

6. Test for Cholesterol (LiebermanBurchard Test) A volume of 0.25 ml CHCl3 was introduced into the 10 drops of eluate. 6 drops of acetic anhydride was then added with 2 drops of concentrated H2SO4 and well mixed. Color was noted. Positive results produced a greenish color which indicated the presence of cholesterol.

7. Test for α- amino acids (Ninhydrin Test) In a clean test tube, 5-8 drops of ninhydrin reagent was introduced into the 10 drops of eluate. It was subjected into heat in the boiling water bath for 15-20 secs. The color produced was observed. Positive results yielded a formation of blue violet color which indicated the presence of α- amino acids.

8. Test for Lipid Unsaturation with Bromine

A volume of 3ml CH2Cl2 was added into 10 drops of eluate was placed in a test

tube. The solution was ensured to be well mixed. 5% of Br2 were added drop wise with CH2Cl2 into the test tube. Solution was shaken after each addition until reddish brown color persisted. Number of drops was noted upon the addition of 5% Br2 in CH2Cl2. Procedure was repeated and result was compared with the following: 8 drops each of coconut, canola, corn and olive oil.

RESULTS AND DISCUSSION: Table 1. Summarized Positive results for each Qualitative Test for Lipids

Chemical Test

Positive Result

Ester

Burgundy color

Glycerol (Acrolein Test)

Burned fat odor

Phosphate

Fine yellow precipitate

Cholesterol (LiebermannBurchard Test)

Greenish color

α- amino acids (Ninhydrin Test)

Blue violet color

Glycerol

Phosphate

Liebermann

Ninhydrin

1st eluate

Yellow solution

No odor

Turbid solution

No color change

Red orange solution

2nd eluate

Yellow solution

No odor

White crystals slightly yellowish

Green

No color

3rd eluate

Burgundy solution

No odor

Turbid yellow with crystals

negative

No color

Chemical Test

Ester

Table 2. Actual Results for each Qualitative Test for Lipids:

Table 1 shows the positive results for each qualitative test performed on lipids while Table 2 shows the actual results for each qualitative performed in lipids. The principles or mechanisms behind each qualitative test are as follows:

Test for Ester Esterification is the general name for a chemical reaction in which two reactants (typically an alcohol and an acid) form an ester as the reaction product. Esters are common in organic chemistry and biological materials, and often have a characteristic pleasant, fruity odor. This leads to their extensive use in the fragrance and flavor industry. Ester bonds are also found in many polymers. Esterification is a reversible reaction. Hydrolysis—literally "water splitting"—involves adding water and a catalyst (commonly NaOH) to an ester to get the sodium salt of the carboxylic acid and alcohol. As a result of this reversibility, many esterification reactions are equilibrium reactions and therefore need to be driven to completion according to Le Chatelier's principle. Esterifications are among the simplest and most often performed organic transformations. The most common esterification processes involve nucleophilic acyl substitution where the carbonyl compound is used as an electrophile and is attacked by a nucleophilic alcohol. However, other processes are possible; esterification by alkylation reverses the roles of "classic" carbonyl chemistry: a carboxylate anion is used as a nucleophile that displaces a halide ion in an SN2 reaction. Acid hydrolysis using sulphuric acid and water (equilibrium reaction). The ester splits into a carboxylic acid and alcohol, protons are donated from the acid. The solution can then be distilled and the remaining acid can be checked using UV indicator. Positive results for the test for ester yields a burgundy color. Based on Table 2, the first and second eluate yielded yellow solution which is a negative result for ester while the third eluate gave a burgundy solution which is a positive result and shows the presence of ester.

Test for Glycerol (Acrolein Test)

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. Based on the results that were culled (Table 2), the first second and third eluate did not produce any odor hence indicates the absence of glycerol for each eluates.

Test for Phosphate The presence of free phosphate in acidic solution can be detected by adding a molybdate to the solution. The equation below illustrates the pertinent reaction between phosphate and ammonium molybdate solution in the presence of nitric acid (HNO3). HPO42-(aq) + 12 MoO42-(aq) + 3 NH4+(aq) + 23 H3O+(aq) -> (NH4)3[P(Mo3O10)4] (yellow)+35 H2O(l) Yellow precipitate results from the reaction in the mixture. When lipids containing phosphate groups in their structures are added to strong acid solution such as the solution used, the lipid hydrolyses, producing free phosphate, forming a yellow precipitate. Based on the results that were culled (Table 2), the first eluate produced a turbid solution, the second eluate produced white crystals which is slightly yellowish and the third eluate produced turbid yellow crystals.

Test for cholesterol (LiebermanBurchard Test) The Lieberman-Burchard or acetic anhydride test is used for the detection of cholesterol. The formation of a green or greenblue color after a few minutes is positive. Lieberman-Burchard is a reagent used in a colorimetric test to detect cholesterol, which gives a deep green color. This color begins as a purplish, pink color and progresses through to a light green then very dark green color. The color is due to the hydroxyl group (-OH) of cholesterol reacting with the reagents and increasing the

conjugation of the un-saturation in the adjacent fused ring. Based on the results that were culled (Table 2), the first and third eluate did not produce any change in color. The second eluate produced a greenish color which indicated the presence of cholesterol.

Test for α- amino acids Ninhydrin Test The principle involved in this test is Oxidative deamination followed by decarboxylation. It is used to detect the presence of α- amino acids. Positive result for this test is Blue-violet solution. Based on the results that were culled (Table 2), the first eluate produced a red orange solution while the second and third eluate did not produced any change in color. The eluates identified based chemical test performed are as follows: 1st eluate: nd

2

on

the

bromine it absorbs. Based on the results that were culled (table 3), The order from the most unsaturated to least unsaturated are as follows: Corn Oil, Olive Oil, Canola Oil, Coconut Oil, 1st 2nd and 3rd eluates (which are on the same level), and finally Vegetable Oil. Possible sources of errors for the experiment were the use of incorrect or wrong reagents and the lack of precision and accuracy in measuring samples or reagents.

REFERENCES: BOOKS: Bettelheim,F.A., March,J. (1990). Introduction to organic and biochemistry. Philadelphia: Saunders College. Heftman, E. (1967). Chromatography. New York: Reinhold Publishing Corporation Lehninger, A.L. (2008). Legninger Principles of Biochemistry. New York: W.H. Freeman.

triglyceride/triacylglycerol

McKee. (2003). Biochemistry: The Molecular Basis of Life. Boston: McGraw-Hill.

eluate: cholesterol

3rd eluate: phospholipid (Lecithin) WEBSITES:

# of drops of bromine

10

10

10

22

71

90

74

Vegetable oil

Olive oil

Corn oil

Canola oil

3rd

2nd

1st

Eluate

Coconut oil

Table 3 Actual Results for Lipid Unsaturation with Bromine:

8

Table 3 shows the actual results for lipid unsaturation with bromine. The test for unsaturation with bromine 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 bromine. The less

Analysis of Lipids Egg Yolk and Milk http://faculty.mansfield.edu/bganong/bioch emistry/liptlc2.htm Retrieved: March 8, 2010 Column Chromatography http://orgchem.colorado.edu/hndbksupport/ colchrom/colchrom.html Retrieved: March 8, 2010

Lipid Library http://lipidlibrary.aocs.org/Lipids/whatlip/in dex.htm#def Retrieved: March 8, 2010