QUALITATIVE TESTS FOR LIPIDS
Lipids found naturally in all living organisms. It has a structural function in the cell, since it presents in
cell membranes, and it is an essential source of energy in the body. It gives more energy than
carbohydrate and proteins. The following tests could be done to differentiate the physical and chemical
properties of lipids.
I. Sudan III Dye Test
Sudan III is a red fat-soluble dye that is utilized in the identification of the presence of lipids,
triglycerides, and lipoproteins.
Principle: Sudan III reacts with the lipids or triglycerides due to similarities in polarity (non-polar). It
will be miscible to fats and oils.
Confirmatory results: reddish orange in color
Lab results: The oil will stain red with Sudan III dye since it is a lipid and contains triglycerides.
However, since the oil is less dense than water and insoluble in water, the oil will form a layer or
globules above the water and appear as a red layer above the water in the test tube.
Samples Sudan III Dye Liquid Detergent LIPIDS -
/+
Water Immiscible (two layers) Liquid detergent acts -
Diluted Honey Solution Immiscible (two layers) as emulsifier due to -
Olive oil Miscible (reddish orange solution) being amphipathic in +
Egg white solution Immiscible (two layers) nature and allows two -
liquids to be miscible
II. Physical Properties of Lipids and Fatty Acids
Lipids are generally insoluble to polar solvents like water but soluble to non-polar solvents like
chloroform. This test determines the degree of solubility of lipids using different solvents.
Principle: same polarity = miscible ; different polarity = immiscible
Solubility of lipids depend on the length of the fatty acids chains and temperature. An
increase in temperature allows the lipids to be more soluble in the given solvent.
Theoretical Lab Results:
Observations
Samples Methylene Chloride Acetone (slightly Ethyl Alcohol (polar)
(polar) polar)
olive oil immiscible miscible immiscible
corn oil immiscible miscible immiscible
animal fat insoluble slightly soluble insoluble
palmitic acid insoluble slightly soluble insoluble
lecithin insoluble slightly soluble insoluble
cholesterol insoluble insoluble insoluble
vitamin E immiscible miscible immiscible
palm oil immiscible miscible immiscible
III. Bromine Test
Bromine test is used to identify the degree of unsaturation in lipids. Unsaturated fatty acids
react to bromine by decolorizing the solution while saturated fatty acids will not exhibit any
change in color.
Principle:
Alkenes react with bromine to give dibrominated compounds. The bromine adds to the
double bond, and one bromine atom is placed on each side of the double bond. The resulting
compound contains only single bond. The addition bromine in a solution containing double bonds
will result to colorless solution. The more double bonds present, the more bromine will be needed
to completely react with the substrate. If the orange color of bromine remains when just one drop
of it is added to a reactant, it means that the bromine did not react with anything and therefore
there are no C=C double bonds present in the substrate.
Confirmatory result:
Colorless = unsaturated bonds
Brown to orange = saturated bonds
Theoretical Lab Results:
Sample Observations
olive oil Decolorizes (presence of unsaturated bonds)
corn oil Decolorizes (presence of unsaturated bonds)
animal fat No reaction (presence of saturated bonds)
palmitic acid No reaction (presence of saturated bonds)
lecithin slightly decolorizes (less unsaturated)
cholesterol No observable change in color (less unsaturated)
vitamin E slightly decolorizes (less unsaturated)
palm oil Decolorizes (presence of unsaturated bonds)
IV. Saponification
Saponification is a process of hydrolysis of oils or fat with alkaline and result in glycerol and salts of
fatty acids (soap). The process of saponification as follows:
The triglyceride (the palm oil) reacts with a strong base (the potassium hydroxide). The KOH breaks
the ester linkages and attaches to each of the 3 fatty acids present in the palm oil structure. The
hydroxide ion (OH-) from the alkali compound (KOH) attaches to the glycerol head of the fatty acid.
The carbonyl group of each fatty acid chain left will be bonded to each of the potassium ions (K+).
The byproduct of this reaction is potassium soaps (potassium palmitate) and glycerol. Soaps made
from potassium hydroxide are liquid while soaps made from sodium hydroxide are hard and solid.
V. Soap Solubility Test
This test shows how metal ions such as calcium, magnesium, lead or iron affects the solubility of
soap in water.
Principle:
Addition of mineral ions causes the deposition of soap and make it insoluble in water, thus
increases its hardness. Hard water is a water containing high mineral contents due to the
deposits of limestones, chalk or gypsum. These mineral ions replace the sodium or potassium
ions present in soap and forms insoluble heavy metal salts. Thus, decreasing the cleansing
property of the soap. Due to the hard water that contain significant quantities of Ca+2, Mg+2 and
some Fe+3, it decreases the foam level of the soap.
Potassium soap + calcium sulfate ===> calcium soap + potassium sulfate.
Confirmatory result:
white precipitates of heavy metals
Theoretical Lab Results:
Observations
5% Calcium chloride 5% Magnesium sulfate 5% Lead acetate
2 mL Soap Solution Formation of Formation of insoluble Formation of insoluble
insoluble white white precipitates dirty white precipitates
precipitates (calcium (magnesium soap) (lead soap)
soap)
VI. Copper Acetate/Sulfate Test
This is used to distinguish a between oil, neutral fat and fatty acids (saturated and unsaturated)
Principle:
The copper acetate solution does not react with the oils (or fats), while saturated and
unsaturated fatty acids react with copper acetate/sulfate to form copper salt.
Copper salt formed in the case of unsaturated fatty acids can only be extracted by petroleum
ether.
Confirmatory results:
Copper salts precipitate
Theoretical Lab Results:
Samples Theoretical Results
Olive oil petroleum ether upper layer containing the dissolved oil and appears
colorless, aqueous solution remains blue in the bottom.
Animal fat petroleum ether upper layer containing the dissolved oil and appears
colorless, aqueous solution remains blue in the bottom.
Corn oil petroleum ether upper layer containing the dissolved oil and appears
colorless, aqueous solution remains blue in the bottom.
Palmitic acid petroleum ether upper layer remains colorless, while consists of
bluish green precipitate of copper palmitate at the bottom.
VII. Liebermann – Burchard Test
This test confirms the presence of cholesterol in the sample. Liebermann - Burchard Test, is a
chemical estimation of cholesterol, the cholesterol is react as a typical alcohol with a strong,
concentrated acids; the product are colored substances.
Principle:
Acetic anhydride is used as solvent and dehydrating agent, and the sulfuric acid is used as
dehydrating and oxidizing agent that causes a change in color. The color is due to the hydroxyl
group (OH-) of cholesterol reacting with the reagents and increasing the conjugation of the
unsaturation in the adjacent fused-ring of the structure.
Confirmatory result:
A positive result is observed when the solution becomes pink to red, then blue, and finally
bluish –green color.
Theoretical Lab Results:
Observations
Chloroform Acetic Anhydride Concentrated H2SO4
Cholesterol The cholesterol will be Dehydrates the OH- Dehydrates the OH-
soluble to chloroform group of the group of the
cholesterol. Formation cholesterol. Formation
of fumes of fumes
References:
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%20conducted%20to,of%20lipids%20in%20a%20solution.
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for-lipids/13050
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