TLC Lipids Lab Report

Vanessa Olga Dagondon

Date Performed:10/06/16

Isabel Palmitos

Date Submitted: 10/17/16 Experiment No. 7

THIN LAYER CHROMATOGRAPHY OF LIPID EXTRACTS I.

INTRODUCTION Lipids are biomolecules that is insoluble or sparingly insoluble in water but soluble

in nonpolar organic solvents. Lipids vary widely on their molecular shapes and size. However, they do not have a common structural figure that would serve as a basis for defining them. The only thing lipids have in common is their insolubility in water (Stoker, 2013). Lipids serve as a major energy source in the body. They play different functions in the body such as providing a hydrophobic barrier that permits partitioning of the aqueous contents of the cells and subcellular structures or maintaining the body’s homeostasis. A disruption on the body’s lipid metabolism may cause some major clinical problems such as atherosclerosis and obesity (Harvey & Ferrier, 2011). Lipids can be classified according to their biochemical function or according to their ability to break down into smaller units through basic hydrolysis (Stoker, 2013). Based on biochemical function, lipids can be grouped into five, (1) Energy-storage lipids (triacylglycerols) (2) Membrane lipids (phospholipids, sphingoglycolipids, and cholesterol) (3) Emulsification lipids (bile acids) 1

(4)Messenger lipids (steroid hormones and eicosanoids) (5)Protective-coating lipids (biological waxes)

On the other hand, based its ability to saponify when it undergoes basic hydrolysis, lipids can be divided as, (1) Saponifiable lipids (triacylglycerols, phospholipids, sphingoglycolipids, and biological waxes) (2) Nonsaponifiable lipids (cholesterol, steroid hormones, bile acids, and eicosanoids) Saponifiable lipids are those that is hydrolysable by heat and alkali while nonsaponifiable lipids are not. Isolation of lipids can be done by using different solvent systems. Two wellknown solvent system for lipid extraction is the Folch and the Bloor solvent system. Folch solvent system consist of chloroform: methanol with a ratio 2:1 while Bloor uses ethanol-ether that has a ratio of (3:1). In order to purify and separate lipid classes, special washes are done with the extracts. Extra care must be considered in extracting lipids since they can easily be oxidized or polymerized. One most convenient separation technique for lipids extract is through chromatographic procedure. TLC or thin layer chromatography is a type of planar chromatography that is considered to be an extremely valuable technique in the laboratory. It provides a rapid separation of compounds that gives an indication of the number and nature of the components of the mixture. It is also used to identify compounds by comparison with standard samples (Christian, 2004). In TLC, separation of mixture is done by allowing a nonpolar (lipophilic) solvent 2

travel upward the plate. The solvent migrate upward the plate through capillary action, carrying the sample mixture along with it. The extent to which the mixture’s components

move along the plate is dependent on their relative affinity to the moving phase (solvent) and the stationary phase (adsorbent). Thus, TLC separation is dependent on the differences in partition coefficients. The ratio of the distance a compound moves to the distant the solvent moves is the Rf value or retention factor. This value is characteristic of the compound, the solvent, and the stationary phase (Christian, 2004). Rf 

dist . traveled by analyte dist . traveled by solvent

By the end of the experiment, students should be able to, (1) Be able to extract lipids from different samples, and (2) Semi-quantitatively and qualitatively identify lipid compositions of the samples through the use of TLC. II. METHODOLOGY Extraction of Lipid Samples Egg yolk, milk, butter, and chicken skin were used as samples for lipid extraction. Approximately 1.0g of each solid sample (egg yolk, butter, chicken skin) was weighed and was placed into a 15mL test tube. 5.0mL ethanol:ether (3:1 v/v) was added into the tube and the solution was centrifuged for about 15 minutes. The nonpolar extract of each sample was obtained and was used for the thin layer separation. For the milk sample, 10mL of evaporated milk was centrifuged for 15 minutes. The separated fat, found as a firm layer on the surface of the skim milk, was transferred into a different test tube and was added with 2.0mL of ethanol:ether (3:1 v/v). Upon addition, the 3 solution was stirred using a stirring rod. The lipids would dissolve and a non-lipid residue would be observed. The nonpolar extract was used for the TLC. -

Thin Layer Chromatography of Extracted Lipids Commercially available silica plate was provided by the laboratory stockroom. Using a pencil and a ruler, seven small dots were drawn in the plate approximately 0.9cm from the edge of the plate. The dots served as a guide during the spotting of the standards and samples. Standard lipid markers (oleic, α-lectin, cholesterol) and samples were spotted into the TLC plate three times (allowing each spot to dry every time) using fine capillary tubes. The TLC plate was placed inside a chamber containing a solvent system of petroleum ether: diethyl ether: glacial acetic acid (90:10:1 v/v/v). The chamber was equilibrated with the same solvent system before use. When the solvent has almost reached to top of the plate, the TLC was removed from the chamber and was allowed to dry. After drying, the plate was placed inside another chamber containing iodine vapor. Once the marks are visible, it was traced with a pencil in order to mark the location of the lipids since it disappears quickly. III. RESULTS AND DISCUSSION Thin Layer Chromatography (TLC) was used in attempt to separate the lipid components of the samples, egg yolk, milk,margarine and chicken skin. The standards used are Oleic acid, α - lecithin and cholesterol. These standards, together with the lipid extracts of the samples, were spotted onto a TLC plate coated with a polar adsorbent, silica and was developed in a chamber containing the solvent system, petroleum ether: diethyl ether: 4

glacial acetic acid (90:10:1 v/v/v). The resulting chromatogram, after exposing to the visualization agent, iodine vapor, is shown in figure 1.

Oleic acid α - lecithin cholesterol milk margarine egg yolk chicken skin

Figure 1. Chromatogram The separated lipids on the chromatogram were marked with a pencil and their corresponding Rf values were calculated. The Rf values for the standards and the lipid extract samples are presented in Tables 1 and 2, respectively. Also Table 2 shows the identity of the separated lipid extracts. Table 1. Rf values of lipid standards Standard Lipids

Distance traveled

Distance traveled

Rf

Oleic acid α - lecithin Cholesterol

by the sample, cm 0.70 2.05 0.35

by the solvent 5.9 5.9 5.9

0.12 0.35 0.06

5

Table 2. Lipid composition of lipid extracts from different sample Sample

Distance

Distance

Rf

Identity

Egg yolk Milk Margarine Chicken skin

traveled by the

traveled by the

sample, cm 2.1 4.1 2.2 2.2 4.7

solvent, cm 5.9 5.9 5.9 5.9 5.9

0.36 0.69 0.37 0.37 0.80

α - lectin Cholesterol α - lectin α - lectin Cholesterol

Before separating the samples into its components using TLC, the samples were first extracted using ethanol:ether (3:1 v/v) solvent system introduced by Bloor. The goal of the extraction lipids is to separate cellular or fluid lipids from other constituents such as proteins, polysaccharides, small molecules (amino acids, etc.) and at the same time, preserve the lipids for TLC analysis. TLC separation brought by the affinity of the samples in the stationary phase and mobile phase. In this case, the stationary phase is the silica embedded on the TLC plate and the mobile phase is the solvent system, petroleum ether: diethyl ether: glacial acetic acid (90:10:1 v/v/v). Silica is very polar stationary phase as shown in its structure in Figure 2.

Figure 2. Structure of Silica: The positive character of the silicon and the negative character of the oxygen make silica a polar stationary phase.

The mobile phase, on the other hand, is a mixture of organic solvents. The choice for the 6 mobile phase is crucial in the separation of the lipids; it is co-dependent on the structure

of the analyte.

In order for the separation to take place, the mobile phase must flow past the stationary phase. This is why the plate was only removed from the chromatographic chamber when the solvent reached the top of the plate. Basically, what happens here is that the lipid components of the sample are drawn into the stationary phase or the mobile phase depending on their affinity on the phases, causing a difference in the distance they travel in the plate. This affinity is based on the old adage, “like-dissolves-like”; polar molecules will have a higher affinity in the more polar stationary phase while the non-polar molecules will have a lower affinity for the stationary phase and will tend to remain in the solvent longer. In this case, the more polar the component lipid in the extract is, the shorter is the distance traveled of that particular component in the plate; the polar functional group in that lipid component tends to interact more with the polar stationary phase causing it to move slower in the plate. Likewise, the less polar the component lipid is, the longer is the distance that it will travel in the plate; the less polar the functional group interacts more with the mobile phase causing it to move faster in the plate. The standards used are oleic acid (Figure 3a), α - lecithin (Figure 3b) and cholesterol (Figure 3c).

(a) (b) 7

(c)

Figure 3. Structures of the standards used: (a) Oleic acid, (b) α - lecithin, (c) cholesterol Oleic acid is a monosaturated omega-9 fatty acid which naturally occurs in some animals and vegetable fats and oils. Fatty acids like oleic acid occur as their esters also known as triacylglycerols. Triacylglycerols are composed a glycerol backbone esterified with 3 fatty acids. Another class of lipid is phospholipids. It is similar to that of triacylglycerols except the a carbon (C3) of the gycerol backbone is esterified to phosphoric acid. Phosphatidic acid is the building blocks of phospholipids. Substitutions, such as choline, can produce phosphatidylcholines also called as lecithin. The third class of lipids is sterols which includes cholesterol and does not contain any fatty acids unlike phospholipids and triacylglycerols which make them non-saponifiable. Based on their structures, phospholipids are the most polar among the classes of lipids mentioned, followed by triacylglycerols and sterols. This means that it is expected for the lecithin to have the shortest distance traveled and also, the lowest Rf value, followed by oleic acid and finally, by cholesterol. However, according to the Rf values obtained, the reverse is shown. This makes the whole data erroneous. This could be explained by experimental errors such not having enough time for development in the chromatographic chamber 8

and by not exposing the chromatogram enough to the visualizing agent, Iodine vapor, especially since the main draw back in using iodine vapor is that it only detects double

bonds, not single bonds, or maybe an error from obtaining the lipid extract by centrifugation. The

samples

were

analyzed

are

the

following:

(1) egg yolk: Egg yolk is composed of 31% of lipids. In this 31%, 65% is neutral lipids which are the triacylglycerols, 30% is phospholipids, and 5% is cholesterol. The bands showing an Rf values of 0.36 and 0.69 may represent oleic acid and cholesterol, respectively. (2) Milk: Milk is composed of 95-96 % triglycerides while sterols such as cholesterol constitute 0.2-0.4% of the total lipid content of milk. The band showing an Rf value of 0.37 may represent low molecular fatty acids. (3) Margarine: Margarine is derived from a combination of plant oils, animal fat and some milk constituents. In contrast, butter is made from milk fat. Margarine contains 80% of fat, 10-20% of which is saturated fatty acids,and relatively high amounts of unsaturated fatty acids. Plant-based margarine has no cholesterol. The band showing an Rf value of 0.37 may represent low molecular fatty acids. (4) Chicken skin: Chicken skin is composed of 75% lipids, 30% of which is saturated fatty acids, 62% is unsaturated fatty acids, and the rest are cholesterol, phospholipids and fatty acid derivatives. The band with Rf value of 0.80 represents cholesterol.

9

IV. CONCLUSION Lipids are biomolecules are insoluble in water but are soluble in organic solvents. They play a vital role in the body as a major source of energy and as a hydrophobic barrier that maintains the body’s homeostasis. Lipids are classified according to their biological functions and their ability to saponify. In this experiment, lipids from different sources are extracted and separated using Thin layer chromatography (TLC). TLC provides a rapid separation of compounds based on their affinity to the mobile and stationary phase. Furthermore, these compounds can be identified by comparison with standard samples. In the experiment, lipids were extracted from egg yolk, milk, margarine and chicken skin using ethanol-ether solvent system. These lipid extracts were separated and identified using TLC with silica embedded on the TLC plate as the polar stationary phase petroleum ether: diethyl ether: glacial acetic acid (90:10:1 v/v/v) as the non-polar mobile phase. In theory, a polar compound to be separated will travel the shortest distance because it would have a higher affinity for the polar stationary phase, while the less polar compounds will travel the longest distance. This can described quantitatively using Rf values. The standards used were oleic acid, a fatty acid which usually occurs in the saponifiable lipids known as triacylglycerols; α - lecithin, a phospholipid similar to that of triacylglycerols except the a carbon (C3) of the gycerol backbone is esterified to phosphoric acid; and, cholesterol which is a sterol, a non-saponifable lipid. These standards can be arranged in increasing polarity: sterols, triacylglycerols, phospholipids. Thus, it is expected for the lecithin to have the shortest distance traveled and also, the 10

lowest Rf value, followed by oleic acid and finally, by cholesterol. Iodine vapor was used as the visualizing agent to enhance the separated bands in the developed chromatogram.

The lipid extracts of the sample showed the following results: for the egg yolk, bands showed an Rf values of 0.36 and 0.69 which may represent triacylglycerols and sterols, respectively; for milk, a band showed an Rf value of 0.37 which may represent low molecular fatty acids; for margarine, a band showed an Rf value of 0.37 which may represent low molecular fatty acids; and for chicken skin, a band with an Rf value of 0.80 represents cholesterol.

V. REFERENCES Akoh, C. C., & Min, D. B. (1998). Food lipids: Chemistry, nutrition, and biotechnology. New York: Marcel Dekker. Thin

Layer

Chromatography.

(n.d.).

Retrieved

October

16,

2016,

from

http://courses.chem.psu.edu/chem36/Experiments/PDF's_for_techniques/TLC.pdf Holtzhauer, M. (2006). Basic methods for the biochemical lab. Berlin: Springer. Steroids - Boundless Open Textbook. (n.d.). Retrieved October 16, 2016, from https://www.boundless.com/biology/textbooks/boundless-biology-textbook/biologicalmacromolecules-3/lipids-55/steroids-301-11434/ Havery, R., Ferrier, D. (2011). Lippincott’s Illustrated Reviews: Biochemistry Fifth Edition. Wolters, Kluwer | Lippincott Williams & Wilkins. Stoker, H. (2013). General, Organic, and Biological Chemistry. Brooks/Cole, Cengage Learning. Christian, G. (2004). Analytical Chemistry Sixth Edition. John Wiley & Sons, Inc. 11

VI. APPENDIX Calculation of Rf values: Rf 

dist . traveled by analyte dist . traveled by solvent

*Oleic acid:

Rf 

*α - lecithin:

*Cholesterol:

*Milk:

Rf 

*Margarine:

0.70cm  0.12 5.9cm

Rf 

2.05cm  0.35 5.9cm

Rf 

0.35cm  0.06 5.9cm

2.2cm  0.37 5.9cm Rf 

2.2cm  0.37 5.9cm

4.1cm  0.69 5.9cm 2.1cm Rf   0.36 5.9cm *Egg yolk: Rf 

*Chicken skin:

12

Rf 

4.7cm  0.80 5.9cm