Isolation and Characterization of Complex Lipids from Egg Yolk

Vince Ivan M. Camangeg, Mark B. Carascal*, Keith Oreil A. Castillejo,

Jasper Lorenz C. Choy
College of Science, University of Santo Tomas, Espaa Boulevard, Manila

ABSTRACT
Several types of lipids are present in egg yolk. These lipids are isolated from the egg yolk through the
Folch method. Furthermore, the isolated lipids were separated from the phosphorylated and
nonphosphorylated components through acetone precipitation. The phosphorylated components are
generally the phospholipids while the triglycerides and cholesterol are the nonphosphorylated
components. Several color reaction tests were used to characterize the components of both the lipid
samples and some standards like cholesterol, lecithin and galactocerebroside. These tests include the
Liebermann-Burchard test and the Salkowski test for cholesterol, Test for phosphate for phospholipids,
Krauts test for choline in lecithin, Ninhydrin test for primary amino group in phospatidylethanolamine and
Molisch test for glycolipids. Overall, the isolation and separation procedures were made possible by the
properties of the lipids in general, and the unique properties of each of its components. Furthermore, the
selective reactivities of the lipid components to different reagents were utilized for the color reaction tests.

INTRODUCTION
Lipids are defined as molecules predominantly consisting of carbon and hydrogen
atoms rendering it nonpolar and generally insoluble in polar solvents (i.e. water). It is a
broad class of biomolecule whose function ranges from being a membrane structural
material, energy storage and messenger molecules. Lipids are categorized into two
main classes based on their structural characteristics and chemical reactivitiessimple
and complex. Simple lipids are esters of fatty acids with various alcohols or simple
chains of fatty acid moieties linked together. Fats and waxes are few examples. On the
other hand, complex lipids are characterized by the presence of additional groups
(aside from alcohol and fatty acid group) to the fatty acid ester. These groups can be
phosphate group, N-bases, carbohydrates, amines, proteins or sulfur groups
(Domodoran, 2011). Common types of complex lipids include phosphoglyceride,

glycolipids and sphingolipids. Lipids are ubiquitous in nature due to its very important
roles. It can be found in every living organism, from simple bacterial cell to complex
animal cells and tissues such as the egg yolk.
Egg yolk is a yellow mass of stored food found in birds or reptiles eggs. Aside from
proteins and carbohydrates, one of its major components is lipids (32-36%). Yolk lipids
are primarily triglycerides (65%), phospholipids (28-30%) and cholesterol (4-5%).
Saturated fatty acids (palmitic, stearic), monounsaturated fatty acids (oleic) and
polyunsaturated fatty acids (linoleic, arachidonic) are the triglyceride components while
the

main

phospholipid

components

include

phosphatidylcholine

and

phosphatidylethanolamine (Mine, 2008). Moreover, glycolipids and hydroxyfatty acids

are also found in the egg yolk. The chemical nature of the components of the egg yolk is
the basis for the technique used in isolating the lipids.
Various methods of separation operate in the principle that lipids are generally soluble
in nonpolar solvents and marginally in polar ones. With this in hand, it is possible to
isolate lipid components from complex samples such as tissues. One of the widely
accepted methods for rapid lipid extraction in tissues is the Folch method which uses a
two-component solvent system containing both polar and nonpolar groups. This method
is still widely used today. On the other hand, separation of the component lipid into
several lipid classes (i.e. simple, complex) can be done by taking into consideration the
reactivities of the various groups present in the lipid. Some of these techniques include
solvent precipitation and chromatography. Lastly, lipids can be detected by the
traditional color reaction tests which operates in the fact that specific groups react with
chemical reagents to produce colored end product.
2

In this experiment, lipid components of egg yolk will be isolated and separation methods
will be used to obtain phosphorylated component and nonphosphorylated ones.
Furthermore, the isolated lipids will be characterized using various color reaction tests.
METHODOLOGY
The experiment was divided into two parts. The first part was involved in the isolation of
lipids from the egg yolk and the subsequent separation of those lipids into its
phosphorylated and nonphosphorylated components. The second part was the
characterization of the complex lipids using various chemical tests. A large-sized regular
white egg was used in the experiment.
A. Isolation of Complex Lipids
An egg was cracked in the evaporating dish and the yolk was carefully
separated. The yolk was then placed in a 250 mL clean beaker and was stirred
together with 100 mL of solvent mixture (a 2:1 by volume mixture of chloroform
[CHCl3] and methanol [CH3OH]). The mixture was then allowed to stand for about
10 minutes. Afterwards, the mixture was filtered through a filter paper and
graduated cylinder was used as the collecting container from which the volume
was noted. Only minimal disturbance on the top solid was observed. The
resulting filtrate was then placed in a clean separatory funnel together with an
equal volume of 1% NaCl solution. The mixture was extracted the separatory
funnel was swirled and the pressure buildup inside it was subsequently released
by opening the vent. After the extraction, the bottom organic layer was
completely collected with a graduated cylinder from which the volume was noted.

The remaining aqueous layer on the separatory funnel was discarded. Then, the
collected organic layer was returned to the separatory funnel with an equal
amount of 1% NaCl and another round of extraction was performed. The bottom
organic layer was collected in 250 mL Erlenmeyer flask. A spatula full of
anhydrous sodium sulfate (Na2SO4) was added to the solution for the purpose of
drying. After which, the solution was filtered and the filtrate was collected in a 50
mL clean Erlenmeyer flask. A pinch of hydroquinone was added to the solution
and was mixed thoroughly. The solution was then transferred to an evaporating
dish which was placed on a steam bath inside the hood. Evaporation was
allowed to occur until the solution was transformed in a sticky yellow residue. The
evaporating dish with the residue was removed from the steam bath and was
allowed to cool on the benchtop for some time. After which, it was placed in an
ice bath and 15 mL of acetone was added and was stirred. The evaporating dish
with residue was cooled in the ice bath for about 15 minutes. Formation of yellow
precipitate was then observed. Afterwards, the acetone solution was carefully
decanted through a filter paper so as to prevent solids from being included in the
decanted liquid. The filtrate was collected in a clean Erlenmeyer flask. On the
other hand, the precipitated residue in the evaporating dish was washed with 5
mL of cold acetone and again was decanted as described earlier. The remaining
residue was added with solvent solution and a pinch of hydroquinone. The
resulting solution was transferred to a clean large test tube, was sealed with cork,
was labeled as PL (phosphorylated lipid) and was refrigerated. All of the acetone
solution collected in the Erlenmeyer flask was evaporated to dryness in the

steam bath inside the hood. After which, the residue on the evaporating dish was
allowed to cool and was dissolved in 3 mL of the solvent mixture (described
earlier). A pinch of hydroquinone was added. The solution was then transferred
to a clean Erlenmeyer flask and was mixed, was sealed with cork, was labeled as
NPL (nonphosphorylated lipid) and was refrigerated.
B. Characterization of Complex Lipids
Six tests were performed for both the lipid samples (PL and NPL) and the
standards (cholesterol, lecithin and galactocerebroside).
B.1 Liebermann-Burchard Test
Half an mL (0.5 mL) of the lipid samples and the standards were placed in five
separate test tubes. Then, ten drops of acetic anhydride were added to each
tube and was swirled gently. Afterwards, four drops of concentrated sulfuric acid
(H2SO4) were added down the side of each tube and were mixed well. The color
of the solution for each tube was noted.
B.2 Salkowski Test
Ten drops of the lipid samples were placed in small test tubes. Then, twenty
drops of concentrated sulfuric acid (H2SO4) were carefully added down the sides
of each tube. Two layers were observed and were not disturbed. The color of the
interphase (the area between the upper layer and the lower layer) were noted.
B.3 Test for Phosphate
One mL of lipid was mixed with the fusion mixture (a 3:1 by volume mixture of
potassium nitrate [KNO3] and sodium carbonate [Na2CO3]) in a crucible. Then,
the crucible with the solution was ignited over a free flame (i.e: Bunsen burner)

until all the organic matter was burned away (i.e. the mixture was turned into
grayish/ colorless solution or into white or gray ash). Afterwards, the crucible with
the product was allowed to cool and was dissolved in 3 mL of warm water. Then,
the contents of the crucible were transferred in a test tube and were acidified with
3M of nitric acid (HNO3). The resulting solution was heated to 65 0C. After which,
3 mL of 2.5 % ammonium molybdate were added and the tubes were warmed
again. The color of the solution and the precipitate formed were observed and
noted.
B.4 Krauts Test
Ten drops of the lipid samples were placed in large test tubes. Then, the tubes
were placed in a boiling water bath inside the fume hood so as to evaporate the
solvent. The resulting dried lipid was suspended in ten drops of distilled water.
Afterwards, fifteen drops of Krauts reagent (diluted bismuth subnitrate in 3M
nitric acid [HNO3] with potassium iodide [KI]) were added. Finally, the tubes were
warmed for 1-2 minutes and the color of the solution and precipitate were noted.
B.5 Ninhydrin Test
Ten drops of lipid samples and five drops of ninhydrin reagent (0.1 g Ninhydrin in
95% ethanol) were placed in small test tubes. The tubes were then warmed in
the hot water bath for 1-2 minutes. Finally, the color of the solution was noted for
each tube.
B.6 Molisch Test
Ten drops of the lipid samples were placed in large test tubes. Then, the tubes
were placed in a boiling water bath inside the fume hood so as to evaporate the

solvent. The resulting dried lipid was suspended in twenty drops of distilled
water. Two drops of Molisch reagent (0.5 g -naphthol in 95% ethanol) were
added and the solution was mixed well. Then, twenty drops of concentrated
sulfuric acid (H2SO4) were added down the side of each tube. Two layers were
observed and were not disturbed. The color of the interphase (the area between
the upper layer and the lower layer) were noted.
RESULTS AND DISCUSSION

Table 1: Observations for the end products of the isolated lipids from egg yolk

Volume of filtrate (organic

layer)
Volume of 1% NaCl used in
extraction
Observation after extraction
(organic layer)
Observation after drying
with anhydrous Na2SO4

77.0 mL
77.0 mL
Turbid yellow solution
Clear, golden yellow solution

Residue (after evaporation)

Sticky yellow

Filtrate (acetone solution)

Clear, golden yellow solution

Phosphorylated lipid

Turbid yellow solution

Nonphosphorylated lipid

Slightly turbid, orange solution with clear

pale yellow solution on top

Table 2: Components of the phosphorylated (PL) and the nonphosphorylated lipids (NPL)

PL

NPL

Phosphatidylcholine (lecithin)

Phospahtidylethanolamine

Triglycerides

Cholesterol

Glycolipids

Table 3: Observations for the different color reaction tests of the different samples; PLphosphorylated lipids, NPL- nonphosphorylated lipids

Samples

Test
PL

Liebermann- Turbid brown

solution
Burchard

Standards
NPL

Turbid dark
green
solution
Turbid pale
brown to
reddish
violet
interphase

Cholesterol

Lecithin

Galactocerebroside

Turbid bluegreen solution

Turbid dark
red- violet
solution

Clear colorless solution

Turbid pale
reddish violet
interphase

Turbid dark
red violet top
solution

Grayish white
interphase

Very pale
yellow clear
solution

NA

Salkowski

Turbid
reddish
brown
interphase

Phosphate

Very pale
yellow, clear
solution with
grayish white
precipitate

Clear yellow
green
solution

NA

Krauts

Red orange
solution with
yellow
orange
precipitate

Clear red
orange
solution with
black
precipitate

Clear red
orange
solution with
black
precipitate

Ninhydrin

Turbid dark
violet
solution

Clear red
orange
solution

Clear
colorless
solution

Molisch

Red orange
interphase

Reddish
violet
interphase

Light blue
green
interphase

Clear red
orange
solution with
yellowish
white-orange
precipitate
Turbid
reddish
brown
solution
Dark red
violet bottom
solution

Light blue-green
interphase

Clear colorless solution

Light green interphase

The isolation procedure done in the experiment is typical of the Folch method as
discussed by Folch et al. (1957). In this method, a 2:1 by volume ratio of chloroform to
methanol was used as a solvent to extract the lipids found in the egg yolk. This solvent
system is generally regarded to extract the most amount of lipid present in biological
samples as compared to other solvent combinations (Folch, et al., 1957; Bligh & Dyer,
1959; Christie, 1993). The properties of each solvent in the system can be accounted
for its ability to separate the lipids from the nonlipid contaminants. Chloroform (CHCl3),
which posses only a weak dipole moment, are only weakly attracted to water and
nonlipid contaminants hence will mostly be in the phase where there is less water and
consequently more lipid. An evidence proved that only an equivalent of 5.5% water can
be solvated in the chloroform phase (Christie, 1993). On the other hand, the more polar
methanol (CH3OH) can attract more water and nonlipid contaminants than the
chloroform and hence can be associated in a single phase. Basically, the isolation
system can be divided into two phases: the methanol phase which contains practically
all the nonlipid substances and a small amount of lipids, and the chloroform phase
which consists essentially of pure lipids from the sample (Folch, et al., 1957; Bligh &
Dyer, 1959; Christie, 1993). Since the main goal of this step is to isolate, at most, the
lipids present in the sample at high efficiency, then the remaining amount of lipids in the
methanol phase should also be isolated. This is accomplished by extracting the sample
mixture with dilute salt solution (i.e. NaCl). This operates in the principle of lipid
distribution altering effect (Folch, et al., 1957). According to this principle, lipids from the
tissue samples, extracted by the mentioned method, exist primarily as salts of sodium,
potassium, calcium or magnesium. In the methanol phase, these salts exist as

dissociated form while it exists undissociated in the chloroform phase. These forms are
in equilibrium with each other in the extraction system. Hence, the addition of salts (i.e.
NaCl) containing cations would decrease the rate of dissociation of lipids due to the shift
in the position of the equilibrium from right to left applying Le Chateliers principle. This
would in turn shift the lipids from the methanol phase to the chloroform phase efficiently.
The mineral salts together with the nonlipid contaminants will remain in the aqueous
layer and can be discarded. On the other hand, the drawback of using the Folch method
is that gangliosides (a class of lipid) is not purified even after washing with dilute salt
solution and will still be included in the aqueous layer (Christie, 1993). Furthermore, the
solvents are also toxic. The final organic layer is composed of lipids, portion of the
solvents and small amounts of water. The water can be removed by adding anhydrous
sodium sulfate (Na2SO4).
Antioxidants (i.e. hydroquinone) are used before further processing of the organic layer.
This retards lipid oxidation (Mrak, et al., 1977) which would otherwise produce byproducts and will give erroneous results in the characterization tests since many lipids
undergo spontaneous oxidation in air. There is also evidence that antioxidants inhibit
lipid peroxidation which would destroy the integrity of the lipid samples (Gavino, et al.,
2000).
On the other hand, acetone precipitation was used to separate phosphorylated (PL) and
nonphosphorylated lipids (NPL) present in the organic layer. Cold acetone is known to
precipitate various types of phospholipids which range from phosphatidylcomplexes and
lecithin (Hills, 1988). It is effective due to the fact that surface active lecithins and
disaturated phosphatidyl complexes are less soluble to cold acetone as compared to
10

other lipid classes. Furthermore, cold acetone induces concentrating effects on those
lipid types which evidently forms the yellow residue (see Table 1). In contrast, sterols
and sphingosines are generally insoluble in acetone (Clark, 1964). Acetone is also used
to eliminate artifacts from storage/ extraction of the lipid samples (Christie, 1993). It is
the solvent of choice as compared to alcohols since it is less reactive to the lipid
components. The composition of the phosphorylated lipids and the nonphosphrylated
lipids can be seen in Table 2.
The six tests employed in this experiment detects for specific groups found in the
different lipid types present in the sample. For instance, the Liebermann-Burchard test
detects for presence of triterpenoids and steroids (i.e. cholesterol) (Krishnaswamy,
2003) specifically sterols with unsaturation in the carbon 5 and carbon 6 of the fused
rings (Cantarow & Schepartz, 1962; Nigam, 2007). Meanwhile, cholesterol is a type of
steroid (a cyclopentanoperhydrophenanthrene derivative) containing hydroxyl group at
carbon 3, unsaturation in carbon 5 and 6 and a hydrocarbon tail in carbon 17 of the 4
fused rings (Lim-Sylianco, 1976). These properties are ideally the characteristics that
Liebermann-Burchard reaction detects. The positive result for this test is the formation
of an initial blue or red solution which was immediately converted to bluish green
solution. This test is also called acetic anhydride-sulfuric acid test due to the reagents
used. Acetic anhydride condenses the hydroxyl group of cholesterol at carbon 3 while
sulfuric acid acts as the dehydrating agent (Clark, 1964). The actual mechanism of the
reaction is not known but it was proposed that the successive evolution of colors was
due to the dehydration, condensation and isomerization reactions (Nigam, 2007;
Sharma & Riyat, 2007). For instance, the unsaturation of cholesterol at carbon 5 and 6
11

is believed to undergo epimerization to produce a 3 form which would subsequently

produce the blue green color. From the data in Table 3, both the cholesterol and NPL
exhibited the positive result. This result is expected for cholesterol as described. On the
other hand, NPL tested positive due to the presence of cholesterol in this component as
seen in Table 2. Similarly, Salkowski test also detects for the presence of sterols (i.e.
cholesterol) in the samples but operates on a different principle as with LiebermannBurchard test. Sulfuric acid (H2SO4), the only reagent used, acts as dehydrating agent
which could either cause two cholesterol molecules to fuse into bisteroids or form
additional unsaturation (Clark, 1964). The reaction can be detected by the formation of
reddish purple interphase. Referring back to Table 3, only NPL and cholesterol were
again positive. As described earlier, this is not surprising for the two samples since it
both contains cholesterol where the reagent selectively reacts. For these two reactions,
the need for perfectly dry glasswares is essential since the reagents are involved in
dehydration and any trace amounts of water can interfere with the results (Sharma &
Riyat, 2007).
The next test involves the detection of phosphate using ammonium molybdate reagent.
The sample with the fusion mixture was first charred to remove the trace organic matter.
Fusion mixture [a 3:1 mixture of potassium nitrate (KNO 3) and sodium carbonate
(Na2CO3)] is a known oxidizing agent (Agarwal, 2000; Srivafava & Jain, 2003) and
facilitates in displacement reactions on certain compounds. Heating process promotes
the reaction and converts some of the trace organic material into CO 2. The resulting
product was then acidified using nitric acid to produce hydrogen phosphate which can
be readily detected by the ammonium molybdate (Beran, 2010). The acidification also
12

hydrolyzes the phosphate group from the fatty acid moiety (although a portion of this
reaction already occurred in the charring process) but its main purpose is to promote
the reaction of the phosphate with that of the ammonium molybdate. Ammonium
molybdate reacts with the acidified solution of hydrogen phosphate by condensation
reaction to produce a canary yellow precipitate of ammonium phosphomolybdate
(Singhal, 2009; Beran, 2010). It should be noted that the rate of the precipitate
formation is in direct relationship with the concentration of the phosphate in the test
solution. Looking at the results in Table 3, only light yellow solution without precipitate
was obtained for both PL and lecithin. This is in contrary to the expected results since
both samples have a phosphate groups as evident with the name phosphorylated
lipids in PL sample and a phosphatidic acid parent for lecithin. A possible source of
error is the quality of the reagents used. Another possible error is the integrity of the
sample because after the acidification process, the samples were stored for a few days
before the processing was done.
On the other hand, Krauts test is used for the detection of choline-containing
compounds. Lecithin, also called phosphatidylcholine, is a phosphatidic acid derivative
with choline (a quaternary amine), stearic acid and oleic acid fatty acid esters (LimSylianco, 1976). The choline group in this lipid forms various quaternary salts or
addition complexes with bismuth potassium iodide (Krauts reagent) (Welcher, 1942;
Cantarow & Schepartz, 1962). The end products (precipitate) are detectable due to its
yellow to orange color which is also indicative of the positive result. The reaction is
similar to that of the Dragendorffs test although it is used primarily for the detection of
alkaloids such as secondary, tertiary and quaternary amines. As seen in Table 3,
13

lecithin and PL both tested positive. This is as expected for lecithin as described earlier.
On the other hand, PL is also positive due to the fact that it contains a particular amount
of lecithin as describe in Table 2.
Ninhydrin test detects for the presence of compounds containing primary amines such
as those found in free amino acids and other compounds like ethanolamine and serine
in lipids (Clark, 1964; Xiao, 2006). The ninhydrin reagent (ninhydrin in 95% ethanol)
decarboxylates the primary amino group to form hydrindantin which would condense to
another molecule of ninhydrin and ammonia to produce the Ruhemanns purple
(Domodoran, 2011). As seen in Table 3, only PL is evidently positive since this lipid
portion contains phosphatidylethanolamine (refer to Table 2). Phospahtidylethanolamine
is a phosphatidic acid derivative containing ethanolamine and fatty acid esters (LimSylianco, 1976).
Lastly, Molisch test is used for the detection of carbohydrates in general. It works on the
principle of the production of furfural derivatives from sugars which subsequently
condenses with -naphthol to give a purple colored interphase (Foulger, 1931). Sulfuric
acid serves as the dehydrating agent while the -naphthol serves as the condensation
agent. As seen in Table 3, only NPL was tested positive. This is due to the fact that NPL
contains the glycolipids present in the egg yolk (refer to Table 2). Glycolipid is a type of
complex lipids which contain various types of carbohydrate attached to the fatty acid
esters. For this, glycolipids are generally positively confirmed by Molisch Test (Chawla,
2003). In contrast, galactocerebroside sample was tested negative. This is somewhat
contrary since this lipid contains galactose (which would have rendered it positive),
sphingosine and fatty acyl group moieties (Lim-Sylianco, 1976). A possible source of
14

error for this is the quality of the sample and the mixing procedure before adding the
sulfuric acid reagent. It is important to mix the solution well before adding the sulfuric
acid so as to distribute proper portion of reactants for the visible color reaction.
CONCLUSION
Based from the experimentation, it can be inferred that lipids from egg yolk can be
isolated

efficiently

nonphopshorylated

via

the

components

Folch
can

method,
be

and

effectively

the

phosphorylated

separated

using

and

acetone

precipitation. Furthermore, it can be inferred that the phosphorylated lipids (PL) of the
egg yolk were tested positive for Phosphate, Krauts and Ninhydrin tests due to the
presence of phosphate group, choline and ethanolamine respectively in this lipid
component. Moreover, nonphosphorylated lipids (NPL) of the egg yolk were tested
positive for Libermann-Burchard, Salkowski and Molisch tests because of the presence
of cholesterol and carbohydrates in this lipid component.
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