Jenalyn D. Osuna

EXTRACTION AND CHROMATOGRAPHY OF LIPIDS I. Introduction Lipids are a major class of biomolecule that is composed of long chains of hydrocarbons and few N- or O-containing functional groups. Some types of lipids may contain polar or charged groups but most of its chemical feature is hydrocarbon-like. This biomolecule is largely insoluble or sparingly soluble in water and soluble in nonpolar organic solvents (Pratt & Cornely, 2014). Some biological functions of lipids includes as an energy source, as an insulation for some vital organs, and as chemical messengers. In this exercise, lipids are extracted from egg yolk using modified Folch method. This isolation method involves the extraction of lipids by adding 2:1 (v/v) methanol:chloroform solvent system. Afterwards, different classes of lipids are separated through silica gel chromatography. Finally, the lipid extracts are extracted through thin-layer chromatography. The objectives of this exercise are (1) to isolate lipids from egg yolk; (2) to obtain the lipid’s components eluted through silica gel chromatography; and (3) to determine the identity of the major lipid fractions from the egg yolk using thin layer chromatography. II. Materials and Methods A. Preparation of Egg Yolk Dispersion A freshly separated egg yolk was used in this experiment. It was diluted with three volumes of 1M NaCl. About 10 mL of the egg yolk stock solution was acquired and extracted with an equal volume of diethyl ether. The sample was centrifuged for 5 minutes at room temperature and then the formed layers were separated. The ether layer was removed by evaporation and the remaining liquid containing the lipids was dispersed by adding same volume of 1 M NaCl. B. Extraction of Lipids from Egg Yolk Under the fumehood, about 1.6 mL of the egg yolk dispersion was mixed with 6 mL of 2:1 (v/v) methanol:chloroform in the centrifuge tube. The contents were mixed by shaking. Afterwards, 2 mL of 1 M NaCl and 2 mL chloroform were added. The tube was cov ered with paraffin and mixed the contents by shaking. The resulting mixture was centrifuged for 5 minutes at room temperature to separate the layers. After centrifugation, the upper layer in the mixture was removed and discarded. The lower layer was transferred to a clean tube and was evaporated under the fumehood. C. Silica Gel Chromatography The silica gel column was already prepared in this exercise. The lipid residue from part B was dissolved in 2 mL petroleum ether. This mixture was poured into the colum n. The eluent or the flow through was collected. The elution was completed by washing the column with 9 mL of a 9:1 (v/v) mixture of petroleum ether and diethyl ether. The eluent was collected in the same tube as the flow through. After the triglyceride elution, 9 mL of 5% methanol in chloroform was added to the column to elute cholesterol fraction. Then, the phospholipid fraction was e luted by adding 4 mL of 1:3:1 (v/v/v) chloroform:methanol:water. D. Thin Layer Chromatography Before the execution of this part of the experiment, 30 mL of solvent system containing 75:25:1 (v/v/v) of petroleum ether, diethyl ether and glacial acetic acid was equilibrated for about 30 minutes. Readily available TLC plate was used for the separation of the components of the samples tested. Using fine glass capillary tubes, the eluents and the standards namely cholesterol, triacy lglycerol and phospholipid were spotted in the TLC plate. The spots are approximately 15 mm apart and 5 mm from the edge of the plate. The chromatogram was developed inside the previously equilibrated chamber containing the solvent system. The TLC plate was removed from the chamber when the solvent has almost reached the top of the plate. Afterwards, the TLC plate was dried. The developed TLC plate was placed and inside a chamber with iodine crystals. The TLC plate was allowed to stand for 5 minutes. Then, the plate was removed from the chamber and the brown spots were marked immediately with a pencil. Their Rf values were determined. Afterwards, the identity of the lipids present in the sample was determined by comparing them with the standard lipids in the TLC plate. III. Results and Discussion The first part of this exercise is the preparation of egg yolk dispersion which involves the addition of 1 M NaCl. This was done to remove water soluble and non-lipid contaminants such as sugar, amino acids and salt that can affect in the yield of the lipids. Lipids extracts have the tendency to trap these contaminants within the lipid micelles (Schneiter & Daum, 2006). The second part was the extraction of the egg dispersion through the addition of 2:1 (v/v) methanol:chloroform. Precipitates were formed after the addition of this solvent system. The addition of the NaCl and chloroform actually formed three layers which is illustrated in Figure 11. The upper layer mainly contains the solvent which evaporates easily while the middle interface contains the cell debris or proteins. These two layers were contaminants and were therefore discarded. On the other hand, the lower layer contains the lipids so that it was saved prior to further processing. The observation during the extraction of the lipid was summarized in Table 11.1. Figure 11.1. Formation of layers in the mixture after addition of NaCl and chloroform Table 11.1. Preparation of egg yolk dispersion and extraction of lipids. Actions / Reagents added Egg yolk  diluted with 1 M NaCl Extract with diethy l ether; centrifugation  organic layer  aqueous layer Egg yolk dispersion Extraction Egg yolk dispersion + 2:1 (MeOH:CHCl3) + 2 mL 1 M NaCl & 2 mL CHCl3 Centrifugation  Supernatant, upper phase (discard)  Residue, lower phase After evaporation Observations yellow colloidal liquid less viscous solution clear liquid yellow liquid yellow mixture formation of white precipitate formation of three layers clear liquid yellow liquid yellow oily precipitate The third part involves the separation of the lipids through silica gel chromatography. Using this type of analysis, the three classes of lipids can be separated by the addition of specific solvent systems in the silica column. This type of chromatographic method employs a stepwise elution. Stepwise elution is associated to an incremental change in solvent in order to help in the development of getting eluents. A series of elution is continued with as many solvents or a mixture of solvents. The arrangement of the solvent being eluted increases polarity or ionic strength (Boyer, 2006). Therefore, the arrangement of solvent mixtures with increasing polarity should be as follows: mixture of petroleum ether and diethy l ether, followed by 5% methanol in chloroform and then, chloroform:methanol:water. After the addition of the first solvent in the column, the triacylglycerols are expected to be eluted. Triacylglycerols (TAGs) are also called triacylglycerides which are fats and oils found in animals and plants. These have acyl groups (R-CO- groups) of three fatty acids that are esterified to the three hydroxyl groups (Pratt & Cornely, 2014). The general structure of TAGs is shown on Figure 11.2. Figure 11.2. General structure of triacylglycerols. After the elution of triglycerides, the addition of the next solvent elutes the steroid compounds. These lipids contain steroid nucleus which is a fused structure of four rings. The steroid nucleus is common feature among steroid compounds. Figure 11.3 shows the general structure of steroids. Figure 11.3. General structure of steroids. Some examples of steroids are cholesterol and testosterone which is shown in Figure 11.4. Cholesterol is one of the most abundant steroids in our body. This is commonly found as the structural components of the cell membranes. On the other hand, testosterone is a kind of steroid hormone associated to manliness and for the maintaining the male secondary sex character. Figure 11.4. Examples of Steroids and their Chemical Structure The last type of lipids eluted in the silica gel chromatography is phospholipids . These type of lipids have polar head group is joined to the hydrophobic moiety by a phosphodiester linkage. The general structure of phospholipids is shown Figure 11.5. Some examples of phospholipids are the given in Figure 11.6. Figure 11.5. General structure of phospholipids. Figure 11.6. Examples of phospholipids and their chemical structures. The mixture of chloroform and methanol has high toxicity which can be hazardous to the indiv idual performing the experiment. In order to address this problem, some non-toxic organic solvents can be employed as solvent system in extracting lipids. One example of non-toxic organic solvent system is the combination of isopropanol and hexane. Isopropanol is a polar solvent that can denature egg proteins and extract polar lipids. Aside from that, it is also soluble in non-polar solvents such as hexane. Isopropanol has low toxicity compared to methanol and chloroform and is therefore accepted as solvent that involves food processes. On the other hand, hexane is the most popular solvent used for the production of v egetable oil. In comparison to isopropanol, hexane will extract simple triglycerides or the neutral lipids. Utilizing the advantages of these to solvents, these are dissolved to make 30:70 ratio of isopropanol and hexane. Increasing the volume of isopropanol compared to hexane will not totally extract most of the neutral lipids (Kovalcuks& Duma, 2014). After the separation of the lipid components, the eluents were characterized by thin-layer chromatography. This was done to distinguish the eluents collected if they are properly separated or not. Three lipid standards namely cholesterol, triacylglycerol and phospholipid were used as basis for comparison. Since the resulting chromatogram has bad resolution, a theoretical chromatogram was provided instead. This theoretical chromatogram is shown on Figure 11.7. Some of the possible errors that interfered with the experimentally prepared chromatogram includes the use of a cracked gel column which may contaminate the eluents, the use of dirty capillary tube that may led to the contamination of the samples being tested and the overflow of the solvent front so that the resulting chromatogram has most spots aligned nearly to the solvent front causing a bad resolution. Figure 11.7. Theoretical chromatogram for egg yolk. On the other hand Table 11.2 summarizes the Rf values and possible identity of the lipid samples from the chromatogram given. The theoretical results in the thin-layer chromatography were analyzed based on their mobility and polarity. Since polar solvents dissolve with another polar solvent, the more polar compound will have lower R f value while non-solvent compounds will have higher Rf value. Non-polar compounds will tend to move with the polar solvent system since they cannot dissolve with polar compounds. Based on the general structure of phospholipid and cholesterol, they are expected to have lower Rf value than the TAG. Both TAG and cholesterol have bulky structure and contain a series of CH bonds making it unreactive to the polar compounds in the solvent system. On the other hand, phospholipids are charged in a solution and therefore it can be dissolved and bind to form iondipole interaction with the solvent system. Table 11.2. Determination of Rf values and identities of lipid samples. Lane Distance Sample Rf No. travelled, mm- Lipid extract- Eluent- Eluent- Eluent- Phospholipid- Cholesterol- Triacyl glycerol- Solvent front- Possible Identity Triacyl glycerol Cholesterol Phospholipid Triacyl glycerol Cholesterol Phospholipid Phospholipid Cholesterol Triacyl glycerol Solvent There are two other ways to visualize the spots from thin-layer chromatography aside from using iodine crystals. One of these is through fluorescence. The silica gel in TLC has the fluorescence material that glows in a UV light. The spots in the TLC plate interferes with the fluorescence of the silica gel so these spots appear as dark spots. The other way is through staining by oxidizing agents such as permanganate and ceric ammonium molybdate. These agents are applied to the TLC plate through spraying or heating. Instead of using thin-layer chromatography in order to analyze the components of the lipids, gas chromatography can be used. The mixture can be separated into its volatile components according to their solubility to the inert material packed in the chromatography column. Although some lipids are naturally volatile, most of the lipid samples in gas chromatography are derived to increase their volatility. For example, in the analysis of phospholipids, the mixture is heated in methanol:HCl or methanol:NaOH. This will convert the fatty acids to glycerol into their methyl esters. In this form, they are loaded into the gas-liquid chromatography column to volatilize the compounds when the column is heated. The fatty acyl esters are most soluble in the column material. Therefore, the less soluble lipids are carried by the inert gas. The order of elution depends on the nature of the adsorbant in the column and the boiling point of the compounds being tested in the lipid mixture (Nelson & Cox, 2005). IV. Sample Calculations 1. Computation of Rf value V. Summary and Conclusion Lipids are biomolecules consisting mostly of long chain hydrocarbons and are mostly insoluble with water. In this exercise, the lipids were isolated from egg yolk by adding 1M NaCl first. This will remove the contaminants that might affect the yield of the desired lipid mixture. Then, 2:1 (v/v) methanol:chloroform was added to totally separate the lipids from other biomolecules present in egg yolk sample. The crude lipid mixture was subjected to silica gel chromatography to separate the three types of lipids namely the cholesterols, triacylglycerols and phospholipids. Through stepwise elution, different solvent systems were added in the column to elute the desired type of lipids. Triacylglycerol were eluted first by adding 9:1 (v/v) mixture of petroleum ether and diethyl ether. Then, cholesterol were eluted by adding 5% methanol in chloroform. Lastly, the phospholipids were eluted using of 1:3:1 (v/v/v) chloroform:methanol:water. The three eluents were analyzed through thin-layer chromatography using 75:25:1 (v/v/v) of petroleum ether, diethyl ether and glacial acetic acid as the solvent system. Each sample including the three lipid standard representing each type of lipids were spotted in the TLC plate letting the solvent flow onto them in the developing chamber. After this, the TLC plate was placed inside the chamber with iodine crystals to view the spots formed. Then, the Rf values were measured for every spot in the chromatogram. Based on the chromatogram, it was found out that the identity of eluents 1, 2 and 3 are triacylglycerol, cholesterol and phospholipid, respectively. VI. References 1. Boyer, R. F. (2006). Biochemistry Laboratory: Modern Theory and Techniques (2nd ed.). New Jersey, United States of America: Pearson Education, Inc. 2. Kovalcuks, A. & Duma, M. (2014). Solvent Extraction of Egg Oil from Liquid Yolk. http://llufb.llu.lv/conference/foodbalt/2014/FoodBalt_Proceedings_-.pdf 3. Nelson, D. & Cox, M. (2005). Lehninger Principles of Biochemistry (4thed.). New York, USA: W.H. Freeman and Company. 4. Pratt, C. &Cornely, K. (2014). Essential Biochemistry (3rded.). USA: John Wiley and Sons, Inc. 5. Schneiter, R. &Daum, G. (2006). Extraction of Yeast Lipids. https://www.unifr.ch/biochem/assets/files/schneiter/publications/LipidExtraction.pdf