Characterization of Lipids ● Consist of 3 fatty acids each in ester linkage with a
Lipids are molecules which are insoluble in H2O (hydrophobic
and amphipathic), a non-structural definition for a primary
biomolecule
● For energy storage (fats & oils)
● Structural elements of biological membranes
(phospholipids & sterols)
● Other functions
○ Enzymes cofactors
○ E- carriers
○ Light absorbing pigments
○ Emulsifying agents in the digestive tract
○ Hormones & intracellular messengers
Simple Lipids
● Include free fatty acids, triacylglycerols, and waxes.
Usually don’t have other functional groups like
phosphates, sulfates, sugars, etc.
● Main function is as energy storage
● Fatty acids - hydrocarbon derivatives at low
oxidation state; carboxylic acids with C4 to C36 long
● Unbranched
● Can be saturated (no double bonds) & unsaturated
(with double bonds)
FATTY ACIDS
● Delta (∆) Notation – begin numbering carbon chain
at carboxyl group
● Omega (𝜔) Notation – begin numbering carbon chain
at tail
TRIACYLGLYCEROLS
● Also known as triglycerides or fats
○ Simple triacylglycerol: Tristearin
○ A mixed triacylglycerol: Stearic
single glycerol
● Insoluble in water and float in water
● Stored in adipocytes or fat cells, serve as energy
storage
● Storage as oils in seeds that provide energy for seed
germination
WAXES
● Esters of long–chain (C14 to C36) saturated and
unsaturated fatty acids with long-chain (C16to C30)
alcohols
● Chief storage form of energy for planktons
● Great water: repellent properties and firm
consistency
● Water fowl: secrete waxes in their feathers
● Other examples
○ Lanolin, beeswax, carnauba wax (palm oil),
spermaceti oil
Complex Lipids
● Include phospholipids, glycolipids, sterols, etc. They
usually contain other functional groups (such as
phosphates, sulfates, sugars, etc.) and have varied
functions such as membrane lipids, act as signals,
pigments and emulsifying agents.
● Membrane lipids are amphipathic; one end of the
molecule is hydrophobic, the other end is hydrophilic
PHOSPHOLIPIDS
● Complex lipids with phosphate group, e.g.
glycerophospholipids or phosphoglycerides
● Consist of two fatty acids attached in ester linkages
to the C1 and C2 of glycerol, and a highly polar or
charged group is attached through a phosphodiester
linkage to C3
● Sample: Lecithin
 STEROIDS
GLYCOLIPIDS
● Complex lipids with sugar functionalities
● Galactolipids - predominate in plant cells; one or
two D-galactose units connected to a glycosidic
linkage to C-3 of glycerol
SPHINGOLIPIDS
● Complex lipids with sphingosine backbone (contain
no glycerol)
● Glycosphingolipids – occur largely in the outer
surface of plasma membranes
● Cerebrosides – a single sugar linked to ceramide
● Found in neural tissues (with D-Galactose)
● Sphingomyelins - contain phosphocholine
or phospho ethanolamine as their polar
head group
● Present in plasma membranes of animal
cells and prominent in myelin
● triterpene derivatives with four fused rings
● STEROLS
○ structural lipids present in the membranes
of most eukaryotic cells
○ consisting of 4 fused rings
○ almost planar and is relatively rigid
○ precursors for variety of products
○ Cholesterol - important component of
biological membranes, and serve as
precursors for bile acids, steroid hormones
and vitamin D
○ Bile acids - polar derivatives of cholesterol
that act as detergents in the intestine,
emulsifying dietary fats to make them more
readily accessible to digestive phase
1. Simple Lipids
a. Saponification test
b. Unsaturation test
c. Emulsification test
d. Iodine test
2. Complex Lipids
 a. Grease spot test Simulation One: Emulsification of Lipids
b. Liebermann-Burchard test
c. Salkowski test Overview: Lipids and water must coexist in the
d. Isolation of Phospholipid from Egg Yolk human body, but they do not readily mix with each other.
e. Tests for Phosphates (Phospholipids and Lipids in liquid form are oils that do not mix with water
Spinghomyelins) because water is polar. Triglycerides are made up of a
three-carbon glycerol molecule with three fatty acid chains
Steps for Lipid Digestion attached to it. Fatty acids are long chains of carbon with
- Digestive enzymes are water-based hydrogen attached, making them nonpolar molecules.
1. Lingual lipase (enzyme) in mouth breaks down large Saturated fatty acids have all single carbon-to-carbon bonds.
fats (triglycerides) Unsaturated fatty acids contain one (monounsaturated) or
2. Additional gastric lipase in stomach is secreted to more (polyunsaturated) double bonds between carbons. An
assist in breaking down since aggregates may form emulsifier is a molecule that can disperse fat in water. A
due to its hydrophobic nature positive result for an emulsifier will mix oil and water better.
3. Once in duodenum, bile salts coat fat droplets and Smaller fat droplet.
cause globules to disperse, creating an emulsion, an
aqueous suspension
4. Due to increased surface area, pancreatic lipase can
hydrolyze triglyceride into free fatty acids and
monoglyceride, which can be absorbed by the small
intestine already.

Isolation of Phospholipid Lecithin from egg yolk. Many

Phospholipids are insoluble in acetone and they precipitate Nonpolar: type of covalent bonding that produces a molecule
out whereas triglycerides, sterol, pigments etc are soluble in without any charge. This is common when many of the same
acetone. Egg and egg yolk is very important sources of or similar atoms are bonded.
lecithin which is a generic term to designate any group of Polar: type of covalent or ionic bond that produces a
yellow-brownish fatty substances occurring in animal and molecule with electrical charges. This is common when atoms
plant tissues, which are amphiphilic – they attract both water that are very dissimilar are bonded.
and fatty substances (and so are both hydrophilic and
lipophilic), and are used for smoothing food textures, Simulation two: Grease Spot Test
dissolving powders (emulsifying agent), homogenizing liquid
mixtures, and repelling sticking materials. Lecithin can easily
be extracted chemically using solvents such as hexane,
ethanol, acetone, petroleum ether, benzene, etc., or
extraction can be done mechanically. It is usually available
from sources such as soybeans, eggs, milk, marine sources,
rapeseed, cottonseed, and sunflower. It has low solubility in
water, but is an excellent emulsifier.

Chemical tests for Phosphate. Phosphates are inorganic salts

containing phosphate ions. It contains a central phosphorus
atom surrounded by four oxygen atoms in a tetrahedral
arrangement. The most well known compound containing
SUDAN IV Test For Lipids
phosphate ions is phosphoric acid. Phosphate ions react with
Lipids are organic molecules that are insoluble in solutions of
ammonium molybdate to produce a characteristic yellow
high polarity like water.
precipitate, ammonium phosphomolybdate. Phospholipids
● Oil and water
and sphingomyelins can give positive results with an
ammonium molybdate test due to the presence of phosphate
Trans fatty acid or trans fat
groups.
● Artificial unsaturated fat
● Contains a trans double bond between carbon atom
 ● Principle
○ Lipids undergo alkaline hydrolysis: releases
glycerol and fatty acids
○ Fatty acids combine w/ either sodium or
potassium ions: form soap
● Reagent
○ Sodium Hydroxide (NaOH) or Potassium
Hydroxide (KOH)
■ Hydroxide has a negative charge to
its point of attack is the one with a
positive charge, the partially
● Saturated fat : butter positive carbon. (will be an
● Unsaturated fat : oil electrophile)
● Trans fat : margarine ○ Ethanol
Fats are made up of glycerol and fatty acids. ● Observation: Presence of bubbles
● Fatty acid chains can be saturated, unsaturated, and ● Indicator: phenolphthalein
trans-unsaturated ● Detects: Free fatty acids
SUDAN-IV ● Low Value: high molecular weight, longer-chain of
● Red stain that detects the presence of lipids. fatty (castor oil)
● Added to a mixture of lipid and water, the dye moves ● High Value: low molecular weight, shorter length
into the lipid layer, giving it a color of reddish-orange (coconut oil)
Hydrogenation ● Will not work on steroids and cholesterol
● Reaction where hydrogen is added across an Unsaturation Test
unsaturated bond (an alkene) ● Known as bromine water test (Halogenation)
● Can be used to change unsaturated fats into ● Principle
saturated fats ○ Presence of alkene and alkyne
○ Detects unsaturated fatty acids or double
Estimation of Saponification Values bonds in a lipid sample
Fats and oils are highly reduced compounds and are ○ ADDITION REACTION: bromine adds to
derivatives of fatty acids. Fatty acids are carboxylic acids with double bond and one Br atom placed on
hydrocarbon chains of 4 to 36 carbons, they can be saturated each side of the double bond
or unsaturated. ● Reagent: Bromine
● Observation: colorless substance
Simple Lipids (E, S, U, I) ○ Vegetable oil, Olive oil, Peanut oil
● Fatty acids (stearic acid in bacon) ○ LECITHIN IS NEGATIVE
● Triacylglycerols (palmitic acid in vegetable oil) ● Detects: Unsaturated fat
● Waxes (beeswax)
Iodine Test
Emulsification Test ● Principle
● Principle ○ Presence of alkene and alkyne
○ Lipids are soluble in alcohol but not in water ○ Unsaturated fatty acids or double bonds in a
● Reagents lipid sample
○ Ethanol ○ Chloroform first
○ Water ○ Di-Halogenation = di-halogenated single
● Observation: Milky white emulsion at top (Fat bond
droplets) ○ Unreacted ICL to form molecular iodine and
○ Many small droplets: presence of emulsifier liberated iodine is titrated with sodium
(+) thiosulfate
● Detects: Generally lipids ○ Potassium iodide solution = pang quantify
how much iodine monochloride
Saponification Test ● Reagent : Iodine (Iodine monochloride)
 ● Indicator: Starch ● Reaction of glycerol to the reagent forms acrolein
● Observation: Colorless substance and two water molecules
● Detects: Unsaturated fat ● Releases a pungent smell if glycerol is present
● Values ● Lecithin & Cooking oil: contains potassium bisulfate
○ Low: less unsaturation (coconut)
○ High: high unsaturation (soybean, sesame, Molisch Test
olive, mustard) ● Detects: carbohydrates (free or bound to lipids or
proteins)
Grease Spot or Spotting Effect ● Detects: glycolipids (lipids connected to a
● Presence of lipids: triglycerides through the carbohydrate)
formation of a translucent or greasy spot on a filter ● Dehydration of sugars to form furfural from pentoses
filter paper or hydroxymethylfurfural from hexoses
● Positive: circle spot Q&A:
● Greasy character of lipids and ability to diffract light 1. When hydrolyzed, complex lipids produce:
that results in a translucent spot ○ Glycerol, 2 fatty acids, phosphoric acid, and
choline
2. Triacylglycerol when hydrolyzed will form:
VEGETABLE OIL NP
○ Glycerol and 3 fatty acids
WATER METHYLENE ETHER TOLUENE 3. Waxes when hydrolyzed
CHLORIDE ○ Long chain fatty acids and alcohol
4. Stearic acid: 18C, Linoleic: 2 double bonds, Linolenic:
Not soluble Not soluble Soluble Soluble
3 double bonds, Palmitic 16C
Polar Slightly Nonpolar Nonpolar 5. Natural form of saturated fat: cis
polar (central (aromatic 6. Shelf life: Unsaturated fats are prone to rancidity
(electronega oxygen hydrocarbon because it is unstable)
tivity of connected ) Binds are
Chlorine) to 2 aryl or nonpolar Iodine
alkyl) between
carbon and Coconut Saturated (Iodine Value lower
hydrogen than 70)
atoms
Soybean Unsaturated

LECITHIN (NP & P) amphiphilic molecule Sesame Unsaturated

WATER METHYLENE ETHER TOLUENE Olive Unsaturated

CHLORIDE (oleic acid, linoleic, linolenic)

Partially Partially Partially Partially Mustard Unsaturated

soluble soluble soluble soluble
Complex Lipids
Polar Slightly Nonpolar Nonpolar
● Phospholipid
L good as an polar
emulsifier ○ Essential structural component of the cell
for a membrane
water/oil ○ Amphipathic
mixture. ● Glycolipid
○ Contains a carbohydrate group
Acrolein Test ○ Regulate cell to cell interactions and signal
● Detects: presence of fat and glycerol transductions
● DEHYDRATION REACTION - water molecules ● Steroid
removed from glycerol by reagent potassium ○ Distinct due to the 4 fused rings in their
bisulfate (KHSO4) and heating the solution structure
○ May contain an -OH group = sterol
 ● Sphingolipid
○ Contains sphingosine backbone Magnesia Mixture Test (for phosphate)
○ Important for proper brain development ● Principle
○ Phosphate reacts with magnesia mixture to
Grease Spot Test form white magnesium ammonium
● Lipids are hydrophobic substances that do not mix phosphate precipitate
well with water ● Reagent
● Light cannot pass through the paper when there is ○ Nitric acid & ammonium molybdate
no liquid in the paper, showing no diffraction at from ● Sample: phosphate salt in aqueous solution
one side to another ● Observation (after a period of time): white
● Milk and Oil is positive precipitate
● A positive result for a lipid will result in an evenly Liebermann-Burchard Test (for cholesterol or steroids)
distributed oily stain ● Principle
Sudan IV Test ○ Cholesterol reacts with acetic anhydride and
● Known as Scarlet Red sulfuric acid resulting to green-blue
● Lipophilic (this is why it is miscible to the lipids) coloration
● Red-colored dye ○ Esterification and Ephemeralization
● If lipid: turns orange ● Reagent
○ Butter, milk, egg, vegetable oil ○ Acetic anhydride
● If not: will form small micelles ○ Concentrated sulfuric acid
Isolation of Phospholipid from Egg Yolk ● Sample: cholesterol
● Reagent ● Observation: green cholesterol
○ Acetone Salkowski’s Test (for cholesterol)
○ 2:1 v/v chloroform and ethanol ● Principle
○ Nitrogen ○ Bi-cholestadiene is formed by the removal
○ Petroleum ether of water molecules from cholesterol by
● Sample: 2 egg yolks sulfuric acid.
○ Contains lecithin that has phosphate group ● Reagents
● Sticky orange substance ○ Concentrated sulfuric acid
● Action ○ Chloroform
○ centrifugation , washing, extraction, and ● Sample: cholesterol
purification of crude lecithin are the key ● Observation: deep cherry-red due to bisulphonic
steps to isolate phospholipid from the egg acid of bi-choestadiene
yolk. ○ The upper layer consists of chloroform in
Dry Heating Test (for phosphate) cherry-red while the acid layer has green
● Principle fluorescence.
○ Heating expands the sample = presence of Kraut’s Test
phosphate ● Reagents: Bismuth subnitrate, 3M HNO3, KI (Kraut’s
● Sample: Phosphate salt reagent)
● Observation (After heating) ● Detects presence of choline
○ Voluminous mass (Swelling of phosphate ○ Sphingomyelin (for brain development) &
salt) Lecithin will test positive
Test for Independent Radicals (for phosphate) ● Positive result: dark orange to red precipitate
● Principle ● Principle involved: complexation reaction
○ Phosphate reacts with ammonium Q&A
molybdate to form deep yellow precipitate Nucleic Acids
● Reagent ● Essential biomolecules found in all living things,
○ Nitric acid and ammonium molybdate including viruses
● Sample: phosphate salt ● Composed of nucleotides
● Observation (after boil heating) : Deep yellow ○ 5-carbon sugar
precipitate w/ ammonium molybdate ○ Phosphate group
 ○ Nitrogenous base
● Basic components
○ Phosphate (=nucleotide)
○ Nucleobase
○ Pentose sugar
● Mononucleotides are linked together by
○ 3’ - 5’ phosphodiester bond
Nitrogenous Bases
● Form nucleosides - components of nucleotides
● 5 nucleobases: A, T, G, U, C
● Fundamental units of genetic code
● RNA - Uracil; DNA - Thymine
Nitrogenous Bases
1. Purine (Adenine and Guanine) 2 rings
2. Pyrimidine (Cytosine, Uracil, Thymine) 1 ring
● Bases are held together by hydrogen bonds
● “A” forms two hydrogen bonds with “T” = A2T
● “G” forms three hydrogen bonds with “C” = G3C
● Specific purine-pyrimidine pairs are due
complementary base pairing
Pentose sugar
1. Ribose sugar
a. Ribonucleic acid (RNA)
i. Uses genetic material to synthesize
proteins
ii. Uracil
iii. single-helix
b. Hydroxyl in C2 position
2. Deoxyribose sugar
a. Deoxyribonucleic acid (DNA)
i. Contains genetic material
ii. Thymine
iii. Double-helix
b. Hydrogen atom in C2 position
c. Termed 2-deoxyribose
Overview of Experiments
1. DNA Isolation
○ Remove other molecules to extract and
isolate pure DNA isolate
2. Hydrolysis
○ Process of breaking the bonds that connect
the components of a nucleotide (Acid or
Enzymatic)
3. Quantitation of DNA using Spectrophotometer
○ Measuring the concentration using
nanodrop spectrophotometer and further
calculating its purity
4. Chemical Characterization of DNA
○ Dische/Diphenylamine test
○ Phosphate test
○ Murexide test
○ Wheeler- Johnson Test
DNA Isolation
1. Place the test tube and the sample in the ice bath
○ Strawberries are octoploids
○ Contain enzymes such as peptidase &
cellulase
2. Transfer the strawberry filtrate to the test tube
3. In a graduated cylinder, dissolve a spoonful of meat
tenderizer into distilled water
4. Add meat tenderizer solution to the strawberry
filtrate
○ Contains proteases or proteolytic enzymes
5. Add ice-cold 95% ethanol in the test tube to
precipitate DNA
○ DNA is least soluble in cold ethanol
6. Spool the precipitated DNA with the glass rod
to Reagents used
1. Detergent: to emulsify membrane lipids and proteins
2. Sodium chloride: neutralizes the negative charge of
DNA that will facilitate precipitation
3. Meat tenderizer: contains protein-digesting enzymes
that will degrade associated proteins that binds the
DNA
4. 95% cold ethanol: to precipitate the DNA from the
filtrate
Materials: test tube, test tube rack, graduated cylinder,
pipette, spoon, glass rod, distilled water
Quantitation of DNA Using Spectrophotometer
● Performed using NanoDrop spectrophotometer
● Beer-Lambert’s Law
○ Linear relationship between absorbance
and concentration
 ● Peaks at 230 nm to 280 nm except 260 nm as it is Determination of DNA Concentration in an Isolate
considered to be impurities
Steps
1. Run a blank through the spectrophotometer using
1.5 uL of the same solution used for diluting the DNA
2. Wipe the measuring area with a clean tissue before
running 1.5 uL of the sample
3. Record absorbances at 260 nm and 280 nm
4. Compute the ratio of A260/A280 to determine the
concentration and purity of the sample

Hydrolysis of DNA
1. Acid Hydrolysis (Total Hydrolysis)
a. DNA is more sensitive in acid hydrolysis
260 nm: Presence of DNA than RNA
● Absorbs UV light due to heterocyclic rings of the b. Total hydrolysis can be achieved by heating
nucleotides, its sugar phosphate backbone does not DNA in 90% formic acid at 180C for 30 min
contribute to this absorption c. The B-glycosidic linkage is selectively
280 nm: Presence of protein cleaved at pH 4, resulting in apurinic sites
● Amino acid tryptophan and tyrosine absorb light at d. DNA is stable at pH 13, only 0.2 of 10^6
this wavelength phosphodiester bonds are broken per
DNA Purity Determination in Abs Ratio minute at 37 degrees
● 1.7 - 2.0 : represents high quality of DNA sample e. Products of acid hydrolysis are phosphoric
● > 2.0: represents “pure RNA” acid, a sugar molecule, and nitrogenous
● < 1.7: represents the presence of protein bases
contaminants

Enzymatic Hydrolysis
● Enzymes that hydrolyze nucleic acids are called
nucleases
● Required for digestion of nucleic acids
○ Exonucleases
■ Cleave at the end of a
polynucleotides (either at 3’ or 5’
end)
○ Endonucleases
■ Cleave at internal site within a
polynucleotide
● Type A: cleaves on 3’ of
phosphodiester bond
● Type B: cleaves on 5’ of
phosphodiester bond
● Restriction Endonucleases
○ Part of the immune system of
microorganisms and are utilized for
protection against foreign DNA
○ Hydrolyze dsDNA at specific internal
sequences
○ Can cleave DNA at specific locations and
combine this DNA fragment in new and
valuable arrangements
● Four types of restriction endonucleases
○ Type 1
■ Require ATP for hydrolysis &
cleaves at random sites within the
DNA with approximately 1,000
base pairs from the recognition
sequences
○ Type 2
■ Does not require ATP for hydrolysis
■ Utilizes energy within the
phosphodiester bonds
■ Cleaves at specific nucleotide
sequences, commonly in
palindromic sequences of dsDNA
○ Type 3
■ requires ATP for hydrolysis
■ Can cleave at specific nucleotide
sequences within approximately 25
base pairs from the recognition
sequences
○ Type 4
■ Targets methylated dna
Chemical Tests for DNA
Steps in Dische/Diphenylamine test
1. Prepare a solution containing banana DNA and water
in a test tube
2. Add the reagents diphenylamine & sulfuric acid into
the solution
3. Place the test tube in boiling water for 10 minutes
4. If DNA is present, the solution should turn blue/black
Test
● Purpose: detection of deoxyribose
● Reagent: Diphenylamine reagent & H2SO4
● Positive result: blue color
● Principle involved: dehydration & complexation
Then
● Deoxyribose undergoes dehydration (H2SO4) and
produces hydroxy levulinic aldehyde, which then
binds with diphenylamine to create a blue complex
 ● The intensity of the blue color is proportional to the 3. Steadily pick up evaporating dish using tongs and
concentration DNA hover over the hot plate
● A clear tube indicates no nucleic acids. A blue color 4. Gently evaporate the solution until a yellow residue
indicates the presence of DNA. A greenish color appears
indicates the presence of RNA 5. If uric acid is present, a strip of red-purple color is
observed after adding a solution of ammonia due to
formation of murexide
Test
● Purpose: detection of purine bases: guanine and
adenine
● Reagent: conc. HNO3; NH4OH
● Positive result: red-purple residue
● Principle
○ Oxidation: reaction of purines with HNO3
yielding dialuric acid and alloxan
○ Condensation: reaction of dialuric acid and
aloozan forming alloxanthin
○ Neutralization: reaction of alloxanthin with
Steps in Phosphate Test ammonia solution yielding murexide
1. Add a small quantity DNA Hydrolysate into the test
tube, then add a small volume of concentrated nitric
acid
2. Boil the contents of the test tube
3. Add ammonium molybdate solution
4. If phosphate is present, formation of yellow
precipitate is observed
Test
● Purpose: detection of phosphate
● Reagent: conc. HNO3; 2.5% (NH4) 2MoO4 solution
● Positive result: yellow precipitate
● Principle involved: precipitation
*Phosphate ions react with conc. HNO3 and ammonium
molybdate to form a yellow precipitate of ammonium
phosphate molybdate

Steps in Wheeler-Johnson Test

1. DNA hydrolyzate is treated with Bromine water
producing a green coloration
2. Adding Barium hydroxide Ba(OH)2 will turn the
solution purple
Test
● Purpose: detection of pyrimidine bases (U & C but
not T)
Steps in Murexide Test ● Reagent: Bromine water, Ba (OH)2
1. Add a few drops of uric acid sodium salt solution to ● Positive result: Purple solution
the evaporating dish ● Principle involved: Neutralization
2. Add concentrated nitric acid to the solution
Observation
● Pyrimidine with bromine water = green coloration
● Through neutralization, Ba(OH)2 will produce dialuric
acid, a purple solution
○ Neutralization: when acid and base react to
form water