Experiment 6
Introduction
Lipids are naturally occurring substances that are arbitrarily grouped together on the basis of
their insolubility in water (a polar solvent) and solubility in nonpolar solvents. Lipids include
a wide variety of different substances, but are commonly subdivided into several classes
based on structural similarities. The examples of classes of lipids are triacylglycerols which is
commonly called as fats and oils, phospholipids which is similar with triacylglycerols but
replace one fatty acid group to a phosphate group and amino alcohol. the last one is steroid
which is lipids that contain the core structure of 17 carbons fused in a ring structure
containing three, six-member rings and one-five member rings.
Procedure
A. Ester Assay
1. Two drops of sample placed in a test tube and dried off the solvent with a gentle stream
of air.
2. The sample was dissolved with 0.5 ml of ethanol and sequentially added two drops each
of 2 M hydroxylamine hydrochloride and of 3.5 M NaOH and mixed well.
3. After heating in a boiling water bath for 30 seconds, two drops of 5 M HCL added and
one drop of 5% ferric chloride in 0.1 M HCL and mixed well.
B. Cholesterol Assay
1. Two drops of sample placed in a test tube and dried off the solvent with a gentle stream
of air.
2. After dissolving the residue with ten drops of methylene chloride, six drops of acetic
anhydride added and two drops of concentrated sulfuric acid and mixed well.
Results
A. Ester Assay
Fraction Observation Result
Triglyceride Magenta colour solution Positive carboxylate ester
Cholesterol Yellow colour solution Negative carboxylate ester
Phospholipids Brown colour solution Negative carboxylate ester
B. Cholesterol Assay
Fraction Observation Result
Triglyceride Green colour solution Positive cholesterol
Cholesterol Colourless solution Negative cholesterol
Phospholipids Dark green colour solution Positive cholesterol
Discussion
Ester assay test is to determine the presence of ester functional group in a solution. This
principle for this test is based on the reaction known as saponification. This reaction is
irreversible because carboxylate ion is inert towards nucleophilic substitution. The result of
this test is the changes of the colour of solution to burgundy or magenta colour for positive
result. All types of lipids in this experiment supposedly get the positive result in this ester
assay test but only cholesterol got the positive result. Triglyceride, cholesterol and
phospholipids contain ester functional group that give the positive result for ester assay. The
result not completely correct as the errors may happened during the experiment.
For the cholesterol assay, the result that we got was negative for cholesterol and positive for
triglyceride and phospholipids. The right result for this test is supposedly positive towards the
cholesterol. The most commonly used assays to identify cholesterol levels that can be
separated into two groups which are gas-liquid chromatography or liquid chromatography
coupled with flame ionization or mass spectrometry detection and quantification. However,
the chromatographic and mass spectrometry methods require specialized equipment and
chemical saponification of cholesterol esters, furthermore, this method require more time and
processing. Those results that we got may wrong because the errors during handling the
experiment or the samples that we got may mix with other substances.
Conclusion
From this experiment, we are able to identify the classes of lipid and the positive and
negative results for the ester assay and cholesterol assay.
References
Usui, S., Hara, Y., Hosaki, S., & Okazaki, M. (2002). A new on-line dual enzymatic
method for simultaneous quantification of cholesterol and triglycerides in
lipoproteins by HPLC. Journal of lipid research, 43(5), 805-814.
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Experiment 7
Introduction
The process for making soap is called saponification. Saponification is a process that involves
conversion of fat or oil into soap and alcohol by the action of heat in presence of aqueous
alkali for example NaOH or KOH. Depending on the nature of the alkali used in their
production, soaps have distinct properties. Sodium hydroxide (NaOH) gives "hard soap".
hard soaps can also be used in water containing Mg, Cl, and Ca salts. By contrast, potassium
soaps, (KOH) are for soft soap.
Procedure
1. 12 ml of NaOH and 27 g of fat were used.
2. The fat warmed in a beaker set into a larger beaker containing water (doublr boiler).
3. NaOH solution was added slowly with steady stirring and took at least 10 minutes for the
brew to thicken up.
4. One or two drops of colouring or fragrance were added and continued stirring until the
stuff piled up when dripped back into the beaker.
5. The mixture poured into small container and let it cooled down in bowl contain of ices.
Discussion
Saponification is an exothermic (release energy) chemical reaction that occurs when fats or
oils (fatty acids) come into contact with lye (a base). Saponification is also an organic
chemical reaction that utilizes an alkali to cleave an ester in to a carboxylic acid and alcohol.
Soaps are sodium salts of of long chain fatty acids. The triglycerides in fats or oils reacts with
aqueous NaOH, it will convert into soap and glycerol. The soap molecule has two parts
which are a polar group (-COO⁻Na⁺) and non-polar group (R-hydrocarbon part). The polar
part also known as head is hydrophilic in nature while non-polar part is tail and hydrophobic
in nature. Depends on the volume of alkali used in soap production, soaps can be divided into
two types, hard soap and soft soap. The mechanisms from the saponification are nucleophilic
attack by the hydroxide, leaving group removal and deprotonation.
The end of this experiment, we were not able to get the soap because not fully heating the fats
before mixing with the alkali solution. The fats not completely melted and rendered. This was
because the chemical called saponification cannot occur between the fats and NaOH which
supposedly result as soap.
Question
1. Why we refer to soap as a “salt”?
Soap refer as salt because consists of potassium or sodium salts of fatty acids obtained
from reaction fatty acid with strong base such as NaOH resulting a salt.
2. What kinds of molecular properties enable substances to dissolve in water?
Water is the best dissolving substance that have polar molecule, negative and positive
charge at each sides.
3. Their guess as to whether the glycerol produced in soluble in water (apparently not
obvious).
Glycerol is easily soluble in water, due to ability of the polyol group to form hydrogen
bonds with the water molecules. Glycerol is slightly denser than water which means
glycerol will sink to the bottom.
4. To show the structures of the products expected from saponification of ethyl acetate.
CH3COOCH2CH3 (ethyl acetate) + NaOH (sodium hydroxide) --> CH3COO-Na+
(sodium acetate) + CH3CH2OH ( ethanol)
Conclusion
From this experiment, we are able to do the saponification process to make the soap and
know about the saponification process.
References
Treub, J. P. (1918). On the Saponification of Fats. I. Koninklijke Nederlandse Akademie
van Wetenschappen Proceedings Series B Physical Sciences, 20, 35-63.
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