Full Report on

Exercise 7

EXTRACTION AND CHARACTERIZATION OF LIPIDS

John Patricia Mae Centeno

CHEM 161.1-3L

2nd Semester AY 2018-2019

Groupmates:
Janelle Allyza Conti

Earlene Lagasca

Jose Lorenzo Manansala

Date performed: 29 March 2019

Date Submitted: 08 April 2019

Aldwin Ralph Briones, RCh.

Laboratory Instructor
 I. Introduction

Lipids are the major source of energy and provide essential nutrients. They are one of the
major constituents of foods, and are important in our diet. Lipids are usually defined as those
components that are soluble in organic solvents such as ether, hexane or chloroform, but are
insoluble in water. This group of substances includes triacylglycercols, diacylglycercols,
monoacylglycercols, free fatty acids, phospholipids, sterols, caretonoids and vitamins A and D.

The lipid fraction of a fatty food therefore contains a complex mixture of different types
of molecule. Triacylglycercols are the major component of most foods, typically making up more
than 95 to 99% of the total lipids present. Triacylglycerols are esters of three fatty acids and a
glycerol molecule. A fatty acid has a carboxyl group at the polar end and a hydrocarbon chain at
the nonpolar tail. Fatty acids are amphipathic compounds because the carboxyl group is
hydrophilic while the hydrocarbon tail is hydrophobic. The carboxyl group can ionize under the
proper conditions. The fatty acids normally found in foods vary in chain length, degree of
unsaturation and position on the glycerol molecule. Consequently, the triacylglycerol fraction itself
consists of a complex mixture of different types of molecules (Campbell and Farrell, 2009). Thus
even a lipid which consists of only triacylglycerols may contain a huge number of different
chemical species. It is often important for food scientists to either know or to be able to specify
the concentration of the different types of lipid molecules present, as well as the total lipid
concentration.

Lipids can sometimes be extracted while its properties are not altered prior to the analysis.
Analysis of the types of lipids present in a food usually requires that the lipid be available in a
fairly pure form. For most foods rigorous extraction methods are needed, such as the solvent or
nonsolvent extraction methods. Once the lipids have been separated they are often melted and
then filtered or centrifuged to remove any extraneous matter (Chapter 3 Lipid extraction
procedures ,1972).

Chromatography can be used to determine the complete profile of molecules present in a

lipid. Thin layer chromatography is used mainly to separate and determine the concentration of
different types of lipid groups in foods, such as triacylglycerols, diacylglycerols,
monoacylglycerols, cholesterol, cholesterol oxides and phospholipids. A TLC plate is coated with
a suitable absorbing material and placed into an appropriate solvent. A small amount of the lipid
sample to be analyzed is spotted onto the TLC plate. With time the solvent moves up the plate
due to capillary forces and separates different lipid fractions on the basis of their affinity for the
absorbing material. At the end of the separation the plate is sprayed with a dye so as to make
the spots visible. By comparing the distance that the spots move with standards of known
composition it is possible to identify the lipids present. Spots can be scraped off and analyzed
further using techniques, such as GC, NMR or mass spectrometry. This procedure is inexpensive
and allows rapid analysis of lipids in fatty foods.

In this exercise, the lipids from the egg yolk was isolated and the lipid components were
separated and the major lipid component in the fractions were determined using qualitative tests
and thin layer chromatography.

II. Methodology

Preparation of Sample Dispersion

The sample of interest in this exercise is the egg yolk which was separated from a freshly
opened egg. Twenty four mL of (2:1) (v/v) dichloromethane-methanol mixture was then added
to the egg yolk. The contents were mixed by shaking and 4 mL of 1 M NaCl and 4 mL of DCM
were then added. The contents were mixed again through shaking prior to centrifugation for 5
minutes at room temperature to separate the phases. The upper phase was removed and
discarded while the lower phase was transferred into another centrifuge tube. The solvent was
evaporated under the fume hood and ample amounts of sample were set aside for the
characterization tests and thin layer chromatography.

Extraction of Lipids from Egg Yolk

Three mL of cold acetone was added into the previous mixture. The contents were then
mixed and placed in the freezer for 15 minutes. The mixture was centrifuged and the qualitative
tests were performed to the residue and centrifugate. The residue was kept and labelled as
“Residue 1” for TLC analysis.
 The acetone was evaporated from the centrifugate and 2.5 mL of ethanol and 10 mL of
saturated ethanolic KOH were added into the mixture. The contents were mixed, covered with
marbles, and then heated in a hot water bath for 15 minutes. The mixture was cooled to room
temperature and was extracted with 3 mL of petroleum ether twice. The organic layer were
combined and the solvent was evaporated in a hot water bath. The residue was set aside and
labelled as “Residue 2” for further analysis. Six M of HCl was the added in the aqueous layer until
precipitate formed. The precipitate was obtained via filtration and the precipitate was labelled as
“Residue 3” and was subjected to further analysis.

Characterization Tests

Salkowski Test

The sample was dissolved in 1 mL chloroform and concentrated sulfuric acid was then added.
The tube was mixed in a vortex mixer to mix the two layers. This was also done for the cholesterol
standard. The upper layer, chloroform layer, should turn red while the lowere layer, sulfuric acid
layer, should turn yellow with green fluorescence.

Reaction with Ammonium Ferrothiocyanate

The sample was dissolved in 2 mL of diethyl ether followed by addition of 2 mL potassium

ferrothiocyanate solution. The mixture was mixed using vortex mixer for 1 minute. This was also
done for the phospholipid standard, lecithin. A red complex would indicate a positive sign of the
reaction.

Saponification
Using a vortex mixer, 0.5 mL sample was mixed thoroughly with 2.5 mL. Followed by addition
of 10 mL 10% sodium hydroxide in ethanol. The mixture was then heated in a boiling water bath
for 15 minutes. After cooling down, the mixture was diluted up to 20 mL. Transferring 5 mL
mixture in two test tubes, 3 mL 6 M HCl was added in the first, while 5 mL 1 M NaCl was added
in the second. This was also done for the triglyceride standard. Appearance of white precipitate
can be observed as a positive sign of reaction.
Thin Layer Chromatography of Extracted Lipids

On the TLC plates, the eluates and standards were spotted using glass capillary tubes.
The order of spotting is as follows: cholesterol standard, tripalmitin (TAG standard), lecithin
(phospholipid standard), crude (egg yolk), residue 1, residue 2, residue 3, and centrifugate 1.
The chromatogram was developed in a solvent system containing petroleum ether, diethyl ether
and glacial acetic acid (75:25:1 v/v) in a chamber previously equilibrated with the same solvent.
The plate was only removed from the chamber when the solvent reached the top of the plate.
Afterwards, the plate was allowed to dry.

To visualize the spots, the developed TLC plate was placed inside a chamber with iodine
crystals for about 5 minutes. When the brown spots were already visible, the plate was removed
from the chamber. The spots were then marked with a pencil immediately.

III. Results and Discussion

Eggs contain proteins of high biological value and other nutrients such as vitamins,
minerals and phospholipids and other lipids. Egg constituents have important functional properties
such as emulsifying, foaming, textural, which make eggs indispensable in many foods, such as
mayonnaise, salad dressings, meringue and bakery products. However, consumption of eggs and
egg products has been affected by concern about dietary cholesterol. Food cholesterol and its
oxidation products, which may appear during dehydrated egg storage are generally believed to
be involved in cardiovascular diseases. Egg cholesterol is found in the yolk. Removal of cholesterol
from the yolk is feasible only if the functional properties, and especially unique emulsifying
activities in products such as mayonnaise and salad dressings, are mostly unaltered. The
extracted lipid material could possibly find use as a fat in human milk substitutes, or as an additive
in cosmetics (Paraskevopoulou and Kiosseoglou, 1994). In this exercise, egg yolk was analyzed
for its lipid components.

Initially, the egg yolk was separated from a freshly opened egg. Twenty-four mL of (2:1)
(v/v) dichloromethane-methanol mixture was then added to the egg yolk. This method was the
same as the Folch method However, all extractions used dichloromethane/methanol (2:1, v/v)
instead of chloroform/methanol. Methanol and chloroform are toxic solvents. Non-toxic solvents
such as tricholotrifluoroethane, isopropyl alcohol, and hexane can also be used to extract lipids
in egg yolk (Cooper, 1977). Folch method is known as the most common solvent system since it
can extract a wide variety of lipids including the neutral and polar ones. Its solvent system is
polar enough to extract all lipids from the source, yet not so polar to extract non-polar lipids.
Dichloromethane is a non-polar solvent used to extract neutral lipids and methanol extracted
polar lipids excluding proteins, nucleic acids and glycogen. Both carbohydrate and amino acids
structures have polar components due to presence of hydroxyl group in the former and nitro
group and corresponding R group in the latter. Only non-polar substances can be dissolved in
non-polar solvents (Nelson & Cox, 2017). Although glycogen is highly polar molecule which can
form hydrogen bonding with polar solvent, it is insoluble in ethanol solution. This is because
glycogen can only interact with the polar side of ethanol which leaves the non-polar region unable
to interact with the rest of the molecule. Solubility of DNA decreases by alcohol in order to
precipitate since alcohols interact with water molecules leaving the DNA molecule interact with
itself. Like DNA molecules, proteins are also insoluble in polar solvents such as alcohol and water.
This is alcohol disrupts the solvation shell created by water molecules precipitating the exposed
proteins (Pace et al, 2004). Separation of the desired component from undesired ones was made
sure by considering the difference in polarity and by using solvent with same polarity as lipid.
Moreover, monophasic system can be obtained by altering the ratio of dichloromethane to
methanol depending on the amount of water in the sample.

The contents were then mixed and 4 mL of 1 M NaCl and 4 mL of DCM were then added.
The membrane of the egg yolk has lipids inside. These lipids and other components of egg yolk
were released upon addition of NaCl solution. The NaCl solution was used for the egg yolk
dispersion to release the lipids in the cells through lysis. The increase in the salt concentration
outside the cell membrane causes it to lyse and release the lipids (Voet and Voet, 2011). The
presence of ionic bridges between the sodium ions and the phosphate group of the phosphoeryl
residues of proteins causes their separation from the egg yolk, leaving other components in the
mixture. As the interaction of acidic lipids to aqueous portions of the egg yolk is prevented, greater
is the lipid yield. This is because of the salt present blocking the acidic lipids from binding with
denatured lipids (Sun, 2016). The contents were mixed again through shaking prior to
centrifugation for 5 minutes at room temperature to separate the phases. The upper phase was
removed and discarded while the lower phase was transferred into another centrifuge tube. The
solvent was evaporated under the fume hood and ample amounts of sample were set aside for
the characterization tests and thin layer chromatography. Observations were tabulated on table
7.1.

Table 7.1. Sample preparation observation.

Actions Taken Observation

Egg yolk Viscous yellow substance

+ DCM:methanol mixture Yellow mixture with dispersed solids

+ 1 M NaCl Yellow mixture with dispersed solids

+ DCM Turbid yellow mixture

Upper Phase White-yellow solids

Centrifugation
Lower Phase Dark yellow liquid

After evaporation of solvent Dark yellow liquid

The lipids was then extracted from the egg yolk by adding 3 mL of cold acetone into the
previous mixture. The contents were then mixed and placed in the freezer for 15 minutes. The
mixture was centrifuged and the qualitative tests were performed to the residue and centrifugate.
The residue was kept and labelled as “Residue 1” for TLC analysis. Acetone was used to separate
the components because it is polar which means its addition will create a biphasic layer wherein
the polar components will mixed with it. Other method can be used to separate the polar and
non-polar components of the lipid mixture such as the column chromatography using silica gel.
Silica gels have very low moisture content, tightest minimal distribution for efficient lipid elution,
minimal impurities and relatively homogenous packing material. On the other hand, alumina and
fluorisil can also be used in column chromatography for lipid elution (Boyer, 2011).

Figure 7.1. Different structures of silanol group in silica surface.

Since the solvent system used in Folch method is composed of polar and non-polar solvents, the
solution of lipids can traverse through the matrix with some retention factors taken into
consideration. As polar lipid, phospolipid elutes last since it is attracted to the polar silanol groups
present at the surface of the silica. Meanwhile, non-polar lipids such as triglyceride and sterols
elute first due to the inability to exhibit intermolecular forces of attraction between the silanol
groups and neutral lipids. Therefore, differentiation between the non-polar components of the
extract will be difficult using this method because they will exhibit the same extent of inability to
interact with silanol groups. While for polar components, the resolution will depend on the polarity
of the structure. The more polar the compound, the longer the time it need to be eluted (Cazes,
2005).

The acetone was evaporated from the centrifugate and 2.5 mL of ethanol and 10 mL of
saturated ethanolic KOH were added into the mixture. The contents were mixed, covered with
marbles, and then heated in a hot water bath for 15 minutes. The mixture was heated to increase
the solubility of KOH. The mixture was cooled to room temperature and was extracted with 3 mL
of petroleum ether twice. The organic layer were combined and the solvent was evaporated in a
hot water bath. The residue was set aside and labelled as “Residue 2” for further analysis. 6 M of
HCl was the added in the aqueous layer until precipitate formed. The precipitate was obtained
via filtration and the precipitate was labelled as “Residue 3” and was subjected to further analysis.
Different solvent systems with varying polarity was used in order to separate the components.
More polar solvent systems are necessary to elute more polar lipids such as phospholipids from
the lipid mixture (Voet and Voet, 2011). Observations were tabulated on table 7.2.
Table 7.2. Observations on isolation of lipids.

Actions Taken Observations

Mixture Dark yellow liquid

+ cold acetone Dark yellow liquid

After placing inside the freezer Dark yellow liquid with dispersed solids

Centrifugation Centrifugate Dark yellow liquid

Residue White solids

After evaporation of solvent in the Dark yellow liquid

centrifugate
+ ethanol Formation of two layers

+ sat’d ethanolic KOH Formation of opaque and transparent yellow layers

Upon heating Distinct two layers

Organic Layer Yellow liquid

Extraction
Aqueous Layer Transparent liquid

Aq Layer + 6 M Little to no ppt formed.

HCl

The next part of the experiment is the characterization tests for the centrifugate and
residues collected. Different qualitative tests were also performed to confirm whether the isolated
compound was the one desired. Salkowski test is for confirmation of cholesterol, reaction with
ammonium ferrothiocyanate for phospholipid and saponification for triglyceride. In the Salkowski
Test, the sample was dissolved in 1 mL chloroform and concentrated sulfuric acid was then added.
As a dehydrating agent, sulfuric acid causes cholesterol fusion resulting to bi-cholestadiene or
formation of unsaturation in the fused rings (Clark, 1964). The tube was mixed in a vortex mixer
to mix the two layers. A positive sign of reaction will be determined when the upper layer or
chloroform layer, turned red while the lower layer, sulfuric acid layer, turn yellow with green
fluorescence. The yellow layer with green fluorescence is a characteristic of bisulfonic acid of bi-
cholestadiene.The crude isolate, centrifugate, residue 1, and the cholesterol standard tested
positive on the reaction.
 Cholestrol Bicholestadiene

Figure 7.2. Salkowski reaction with cholesterol.

The next test is the reaction of samples with potassium ferrothiocyanate. The sample was
dissolved in 2 mL of diethyl ether followed by addition of 2 mL potassium ferrothiocyanate
solution. The mixture was mixed using vortex mixer for 1 minute. This was also done for the
phospholipid standard, lecithin. A red complex would indicate a positive sign of the reaction. The
red inorganic compound potassium ferrothiocyanate is insoluble in chloroform but forms a
complex with dipalmitoyl lecithin which is freely soluble in chloroform. When a solution of
chloroform containing dipalmitoyl lecithin is mixed intimately with ammonium ferrothiocyanate at
room temperature, a colored complex is formed which partitions in the chloroform phase
(Stewart, 1980). The crude isolate, centrifugate, residue 1, and the phospholipid standard lecithin
tested positive on the reaction.

Lastly, the samples underwent saponification. Formation of precipitate or cloudy mixture

can be observed as a positive sign of reaction. Using a vortex mixer, 0.5 mL sample was mixed
thoroughly with 2.5 mL. Followed by addition of 10 mL 10% sodium hydroxide in ethanol. The
mixture was then heated in a boiling water bath for 15 minutes. After cooling down, the mixture
was diluted up to 20 mL. Transferring 5 mL mixture in two test tubes, 3 mL 6 M HCl was added
in the first, while 5 mL 1 M NaCl was added in the second. This was also done for the triglyceride
standard. In presence of heat, fatty acids react with strong bases such as sodium hydroxide and
potassium hydroxide yielding white precipitate of soap in water. Residue 1, TAG standard
tripalmitin, crude isolate, and centrifugate 1 tested positive on the reaction. Observations are
tabulated on tables 7.3, 4, and 5 and the summary of results are tabulated in table 7.6.
 Figure 7.3 Saponification of fatty acids using strong bases.

Table 7.3. Observations on Salkowski Test.

Sample Observations

Residue 1 Formation of red top layer and yellow bottom layer with green
fluorescence
Residue 2 Clear top layer, yellow bottom layer

Residue 3 Colorless liquid

Cholesterol Standard Formation of red top layer and yellow bottom layer with green
fluorescence
Crude Isolate Formation of red top layer and yellow bottom layer with green
fluorescence
Centrifugate 1 Formation of red top layer and yellow bottom layer with green
fluorescence
Table 7.4. Observation. on the reaction of samples with potassium ferrocyanate.

Sample Observations

Residue 1 Single layer, red solution

Residue 2 Red top layer, colorless bottom layer

Residue 3 Red top layer, colorless bottom layer

Lecithin Single layer, red solution

Crude Isolate Single layer, red solution

Centrifugate 1 Red top layer, colorless bottom layer

Table 7.5. Observations on the saponification.

Observations
Sample
6 M HCl 1 M NaCl

Residue 1 Formation of ppt Formation of ppt

Residue 2 Clear liquid Clear liquid

Tripalmitin Formation of ppt Formation of ppt

Crude Isolate White cloudy mixture White cloudy mixture

Centrifugate 1 White cloudy mixture White cloudy mixture

Table 7.6. Summary of the results of different chemical tests.

Observation

Sample Reaction with potassium Saponification

Salkowksi Test
ferrocyanate Add’n of Add’n of
HCl NaCL
Residue 1 + + + +

Residue 2 - - - -

Residue 3 - -

Cholesterol +
Standard
Phospholipid +
Standard
TAG standard + +

Crude Isolate + + + +

Centrifugate 1 + - + +

Based on the result of the chemical tests, the residue 1 possibly contains cholesterol,
phospholipds, and TAGs as it gave positive results in all tests. The residue 2, however, did not
react on any chemical tests. The residue 3 was expected to be TAG. Possible source of error may
include the improper separation of the lipid components of the egg yolk that caused impurities in
the residue collected.

The next part of the experiment is the thin layer chromatography. The mobile phase is
the relatively nonpolar solvent system which is the petroleum ether:diethyl ether:glacial acetic
acid mixture. The glacial acetic acid was one of the components to give a slightly polar nature of
the solvent for the polar stationary phase, but generally, the mobile phase is nonpolar in nature.
On the other hand, the stationary phase is the polar silica gel. Silica gel or the Kieselgel is the
most frequently used stationary phase or adsorbent in thin layer chromatography lipid analyses.
The required particle size used for standard TLC lipid analyses ranges from 10 to 15 micrometers
with pores of 60 Å in diameter (Silica Gel 60) (Boyer, 2011)

Fine glass capillary tubes were used for spotting the sample and standards in the thin
layer chromatography plate. The groups labeled the possible lipid components and the spots of
the samples for proper references. After spotting the standards and the samples, it was allowed
to be developed in a solvent system containing the solvent system for this experiment. It was
done inside a glass chamber previously equilibrated with the same solvent.

After the solvent reached the top of the plate, the TLC plate was then removed from the
developing chamber and was allowed to dry. The developed TLC plate was then placed inside a
chamber with iodine crystals. This nonspecific stain was used so that one can visualize the
progress of separation. Lipids are generally colorless; thus, need to be detected and visualized
using proper chemical reagents.

Figure 7.4. Structures of triacylglycerol (left), phospholipids (right, a), cholesterol (right, b).

There are three main types of associations in which lipids participate: Van der Waals or
hydrophobic association in which ‘neutral’ or non-polar lipids, such as sterol esters, glycerides,
hydrocarbons, and carotenoids, are bound by relatively weak noncovalent forces through their
hydrocarbon chains to other lipids and to hydrophobic regions of proteins; examples are, fat in
adipose tissue, chylomicrons, albumin-fatty acid complexes, oil droplet inclusions in microbial
cells; the next on is the hydrogen bonding which is the electrostatic and hydrophobic association
in which polar lipids, such as phosphatides, glycolipids, are bound to proteins by hydrogen
bonding, electrostatic or hydrophobic forces, as in plasma membranes, mitochondria,
endoplasmic reticulum, and serum lipoprotein complexes and lastly the covalent association in
which fatty acids, hydroxy acids, or complex branched acids are linked covalently as esters,
amides or glycosides to polysaccharide structures, as in lipopolysaccharides of bacterial cell walls
(Chapter 3 Lipid extraction procedures, 1972).

Figure 7.5. The structures of the lipid standards, (a) phospholipid standard, (b) cholesterol
standard, (c) triglyceride standard.

Shown in Figure 7.5 are the structures of the lipid samples in the experiment. Based on the
structures of some common lipids, it can be deduced that the most polar lipid are the
phospholipids. Phospholipids should travel the least distance with a polar stationary phase
compared to the others. Phospholipids contain the phosphate groups as well as the esters in the
glycerol making it the most polar. The phosphate group and the ester groups are capable of H-
bonding. Phospholipids are the major component of cell membranes. Examples of phospholipids
are Phosphatidic acid (phosphatidate) and Phosphatidylethanolamine (cephalin). Most common
sources of phospholipids are soya, rapeseed, sunflower, chicken eggs (Nelson and Cox, 2017).

The next more polar would the triacylglycerides due to the presence of the three ester
groups in the chains. These ester groups are also capable of H-bonding. Examples of TAGs include
palmitic acid, oleic acid and alpha-linolenic acid. Dietary sources of TAGs are fishes, beef, olives
and coconut (Nelson and Cox, 2017).

The most non-polar would be the steroids like cholesterols which are composed of four
fused-rings. The presence of the rings contribute to its overall non-polarity and compactness.
Steroids contains only one polar group (hydroxyl) compared to the other lipids making it the least
polar among them. Examples of steroids are cholesterols, testosterone and estrogen. Dietary
sources of cholesterol are cheese, egg yolks, beef and pork (Voet and Voet, 2011). The sequence
of their polarities is:

PHOSPHOLIPID > TRIACYLGLYCERIDES> STEROIDS

(Most Polar Lipid) (Least Polar Lipid)

The chromatogram is shown in figure 7.6. The Rf values were determined in the TLC. Table 7.7
shows the Rf values in the thin-layer chromatography.

Figure 7.4. Thin Layer Chromatogram.

Table 7.7. The values of the standards and samples for lipid thin layer chromatography.

Solvent Distance travelled, mm Rf

Solvent front 80.586

Lecithin 60.286, 34.294, 22.548, 0.748, 0.426, 0.280, 0.212

17.118
Cholesterol standard 25.323 0.314

Tripalmitin

Crude 62.804, 33.350, 25.146, 0.779, 0.414, 0.312,

22.725, 17.472 0.281, 0.217
Residue 1 33.291, 25.381 0.413, 0.315

Residue 2 35.655, 25.35 0.442, 0.314

Residue 3

Centrifugate 1 61.270 0.760

Based on the results, it can be seen that the chromatogram developed well. It can be
seen that the crude lipid produced multiple spots which could illustrate that it contains all three
lipid components. It can be concluded that the crude isolate contains different lipids as seen on
its Rf values which are close to lecithin, tripamitin, and cholesterol standard. Residues 1 and 2
are potentially composed phospholipid and cholesterol. The centrifugate 1 possibly contains
phospholipid. Tripalmitin, however, did not show development in the chromatogram since it is
highly non-polar, hence, it did not bind in the asorbent.

The iodine vapor is the visualizing agent for the lipid component separation in the thin
layer chromatographic plate. It reacts with the double bonds of the lipid to form a reversible
reaction. Beside iodine crystals as the visualizing agent for lipids, other reagents can also be used
for effective viewing of the separated lipids using thin layer chromatography. Coomassie blue
R250 can be also used for visualizations in lipid classification. The use of UV light is one of the
visualization techniques that can be employed. This technique just uses a simple fluorescent dye
or any staining reagent for it to be seen under the UV light. Sulfuric acid can also be employed.
In this case, this reagent is just sprayed or just placed in minute amounts on the chromatogram
itself. Afterwards, it should be heated at a certain, relatively average temperature, and then the
compounds present would form dark-colored spots. This must be viewed on a clear white
background for effective viewing (Boyer, 2011).

Compared to thin layer chromatography, gas chromatography (GC) can only analyze
volatile substances or substances that can be derivatized or converted into volatile forms. Intact
triacylglycerols, free fatty acids, and cholesterol are not very volatile in nature and are therefore
not recommended for analysis using GC. However, derivatization of lipids can be done prior to
analysis to increase their volatility. Triacylglycerols are first saponified which breaks down to
glycerol and free fatty acids, and are then methylated. Saponification reduces the molecular
weight and methylation reduces the polarity, both of which increase the volatility of the lipids
(Voet and Voet, 2011).

Lipids are fats providing energy in hormonal production and for muscle and body
processes. Foods cannot be digested and absorbed properly without lipids. Lipids produce bile
acids in liver to allow mixing fat and water in intestines. Hence, lipids provide about half of the
fuel that body needs when at rest or during everyday activity and they are stored as adipose
cells. It also transports fat-soluble vitamins such as vitamins A, D, E and K fro intestines to blood
stream. Aside from being energy source, lipids also insulate and protect the body. The internal
body temperature is kept regular despite of the external temperature because of the fat layer
just below the skin. Organs are also protected by a fat layer around them acting like a bubble
wrap so bump and bruise caanot hurt them (Kannall, 2018). Moreover, essential lipid are not
produced by the body and are only supplied by the diet. As a response, food industries utilize
lipids in production od cookies, crackers, cakes, muffins, pie crusts, pizza dough, breads,
margarine, baking and drink mixes, hard taco shells, candies and frozen dinners. Trans fats
provide crispy texture and increase shelf life of the food. Unfortunately, there are diseases coming
from saturated and trans fats such as cardiovascular diseases. There are also lipid metabolism
disorders such as Gaucher diseases and Tay-Sachs disease.

IV. Sample Calculations

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑒𝑙𝑢𝑒𝑛𝑡 (𝑚𝑚) 60.286 𝑚𝑚

𝑅𝑓 = = = 0.7480952
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑡𝑟𝑎𝑐𝑘𝑖𝑛𝑔 𝑑𝑦𝑒 (𝑚𝑚) 80.586 𝑚𝑚
 V. Summary and Conclusion

In the experiment, the lipids from the egg yolk was separated based on extraction using
dichloromethane:methanol solvent system. Next, the lipid fractions were separated using
different solvent systems on the basis of the different polarities of the lipids. Then, the thin layer
chromatography was employed for the identification of the lipid fraction based on its polarity and
its affinity towards both the stationary phase and mobile phase.

Based on the Rf values, it was found out that phospholipids had higher Rf values than
cholesterols, meanwhile, triglycerides were not detected on the TLC plate. Ideally, triglycerides
should have a lower Rf value than cholesterols based on their polarities. The Rf values of the lipid
fractions correspond closely with the Rf values of the lipid standards.

Overall, the experiment was successful. Lipids were isolated from egg yolk, and the lipid
components were separated using the solvent extractions. The identity of the major lipid fractions
were identified using the thin layer chromatography qualitative tests. Residue 1, Crude isolate,
and centrifugate 1 tested positive for Salkowski test which means that these samples contain
cholesterol, residue 1 and crude isolate tested positive in reaction with ammonium
ferrothiocyanate which means they contain phospholipids and residue 1, crude isolate, and
centrifugate 1 tested positive in the saponification which means they contain TAGs.

VI. References/ Literature Cited

Boyer, R. (2011) Biochemistry Laboratory: Modern Theory and Techniques 2nd Edition. New
York: Prentice Hall.

Campbell, M.K., Farrell, S.O. (2009) Biochemistry, 6th ed. California: Thomson Brooks/Cole.

Cazes, J. 2005. Encyclopedia of chromatography. USA: CRC Press

Chapter 3 Lipid extraction procedures. (1972) Laboratory Techniques in Biochemistry and

Molecular Biology, 347–353. doi:10.1016/s0075-7535(08)70549-7

Cooper, T. G. (1977) The Tools f Biochemistry. New York: John Wiley & Sons, Inc.

Kannall, E. 2018. What are lipids used for in the body? Retrieved from
https://healthyeating.sfgate.com/lipids-used-body-8282.html
Nelson, D. L., Lehninger, A. L., & Cox, M. M. (2017). Lehninger Principles of biochemistry.
Macmillan Higher Education: Basingstoke.

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