EXPERIMENT 6: LIPIDS
is cis- (naturally occurring)
Lipid
 Water insoluble biomolecule not
associated into polymers
 Soluble in fat solvents: CHCl3, ether,
C6H6
 Either hydrophobic or amphipathic, tend
to form surface monolayer, bilayer,
micelles or vesicles when in contact with
water
Function
 Major component of biomolecules
 Storage and transport form of energy;
alternative source of energy
 Regulatory molecule
 Thermal insulator
 Extra- and intracellular signaling
molecules
Lipid Type
1. Fatty Acids*
 Long chain monocarboxylic acids
(pKa = 4.5 -5.0)
 Have long hydrophobic tail and a
polar head (amphipathic)
 Differ from one another in:
 Length of hydrocarbons tails
 Degree of saturation
 Position of double bonds
 Types:
a.) Saturated
- CnH2n+1COOH
- Without unsaturation/ C=C
- waxy solids at room
temperature (animal fats)
b.) Unsaturated
- CnH2n-2COOH, CnH2n-
3COOH, CnH2n-5COOH
- Fatty acids with one or more
olefinic functionalities
- liquids at room temperature
(vegetable fats)
- Orientation at the double bonds
- Inserts a bend in the HC chain =
contribute to membrane fluidity
Physical Properties of Fatty Acids
Solubility – generally water-insoluble
 Soluble in non-polar organic solvent
 As number of Cs increases, solubility
in organic solvent increases
Melting and Boiling Point
 As number of Cs increases, MP and
BP also increase
 Presence of unsaturation decrease
MP
Reactions of Fatty Acids
 Ester Formation
 Addition reaction (for olefins)
A. Hydrogenation
B. Halogenation
 Oxidation
A. Dilute KMNO4 (mild oxidation)
B. Rancidification – slow and
spontaneous reaction of double
bonds with atmospheric O2 or with
certain oxygenases to form short
chain aldehydes responsible for
objectionable odor and unpleasant
taste
C. Ozonolysis – reaction of double bond
w/ ozone (O3) to form aldehydes and
carboxylic acids
2. Saponifiable Lipids*
A. Triacylglycerols
 Storage form of fatty acids
 Can be solids (fats) or liquids
(oils)
 Triesters of fatty acids and
glycerol
 Can be SIMPLE (the three f.a.
units are the same) or MIXED
(the three f.a. differ)
Reactions of Triacyglycerols
  Saponification – alkaline
hydrolysis if fats and oils yield
soap and glycerol
 Addition reactions
 Oxidation reactions
B. Glycerophospholipids
 Phosphoglycerides or
Glycerophosphatides
 2 fatty acids + glycerol + H3PO4
+ group X
 Most abundant lipid membranes
 Glycerol backbone
 α2 – usually esterified to LCSFA
(16:0, 18:0)
 β – esterified to unsaturated fatty
acid (18:1, 18:2, 18:3, 21:4)
 amphipathic and amphoteric
molecules
 Cardiolipins
(Diphosphatidylglycerol) –
abundant in cell membranes of
mitochondria and chloroplast
 Lecithins (Phosphatidylcholine) –
most abundant phospholipid in
membranes (50%); alcohol
soluble, acetone-insoluble tissue
extracts; surface active agent or
emulsifying agent; mild H+
hydrolysis, free f.a. +
glycerophosphate + choline
 Strong hydrolysis: free f.a. +
glycerol + H3PO4 + choline
C. Sphingolipids
 Abundant in tissues of the CNS
 Backbone molecule: Sphingosine
(D-erythro-1,3-dihydroxy-2-
amino-4-trans-octadecene)
(trans-4-sphingenine)
D. Glycoglycerolipids
 Abundant in chloroplast of plants
and archaeabacterial membrane
E. Waxes
 Non-polar esters of long chain
fatty acids anf long chain
monohydroxylic alcohols
 Completely insoluble in water,
functioning as water repellants
 Feathers of birds, leaves of
plants
3. Non-saponifiable Lipids
A. Eicosanoids
 Prostaglandins – have
cyclopentane ring and two side
chains w/ a –COOH; functions:
control of blood pressure,
stimulate muscle contraction,
induce inflammation, inhibit
platelet aggregation
 Thromboxanes – promote platelet
aggregation and smooth muscle
contraction (vasoconstriction)
 Leukotrienes – promote
constriction of smooth muscles
(bronchoconstriction)
B. Steroids
 Non-saponifiable fraction of lipid
extracts
 Derivatoves of tetracyclic
hydrocarbon
cyclopentanoperhydrophenanthre
ne
b. Sterols
 With –OH at C3 of the ring
 Cholesterol – precursor for
the synthesis of other
steroid; derived from
squalene, a C30 terpenoid
HC: wealdy amphipathic;
C3 – OH can be esterified
to form Cholesteryl esters
which are hydrolysate;
bulky and rigid and fits into
membrane lipids disrupting
membrane regularity
 Bile acids – emulsify
dietary lipids in the
 intestine; secreted by the
liver and stored in the gall
bladder; most abundant in
humans cholic acid;
chenodeoxycholic acid
 Steroid Hormones
i. Progestins –
progesterone
ii. Androgens –
androstenedione,
testosterol
iii. Estrogens –
estrone, estradiol
iv. Glucocorticoids –
cortisol,
corticosterone
v. Mineralcorticoids –
aldosterone
C. Lipid-soluble Vitamins
 Vitamin D – most abundant
form is D3 (cholecalciferol)
 Vitamin A (trans-retinol) –
isoprenoid alcohol that plays a
key role in vision, control of
animal growth & stimulation of
development of nervous
system, can either be
consumed in diet (cod liver oil,
fish livers, butter, eggs) or
biosynthesize from β –
carotene
 Vitamin E (α-tocopherol) –
has an antioxidant role =
prevents attack of peroxides
on unsaturated f.a. in
membrane lipids
 Vitamin K – important in the
lymphatic synthesis of
prothrombin and protein
factors in blood platelets;
Vitamin K1 (phylloquinone)
found in plants; Vitamin K2
(menaquinone) found largely
in animals and bacteria
D. Terpenes
 Generic name for all compounds
biosynthesized from isoprene
precursors
 Includes terpene HC, alcohols,
aldehydes, and terpenoid
vitamins
 Insect and plant growth
hormones, plant’s pleasant
odors, lipid-like sugar carriers
Egg Yolk
 Rich source of a variety of biochemically
important compounds such as proteins
and lipids (glycerides or fats,
cholesterol, cholesterol esters, and
phospholipids)
 Egg lipids may be divided into two
classes:
1. Non-phosphorylated lipids
 include cholesterol, cholesterol
esters and triglycerides
2. Phospholipids (those containing a
phosphate entity)
 Also known as
glycerophosphatides
 Widespread and occur in all plant
and animal cells as major
structural components of cell
membranes
 They play critical roles in the
transport of molecules across
membranes, storages and
metabolism of fatty acids, and as
activators in the blood clotting
process
Characterization of Lipids
A. Test for Sterols
1.) Salkowski Test
 Reagents: conc. H2SO4, CHCl3
 + result: cherry red solution
 Principle:
1. Addition reaction
 2. Condensation to form a
bisteroid
2.) Liebermann-Burchard Test
 Reagents: conc. H2SO4, acetic
anhydride
 + result: emerald green color
 Principle: esterification of C3 –
OH and epimerization of C5
double bond
* a brown color indicates the
absence of cholesterol
B. Test for Glycerophosphatides and
Sphingolipids
1.) Acrolein Test
 Reagents: KHSO4
 + result: acrid, irritating/ burnt fat
odor
 Principle: dehydration and
oxidation
2.) Molisch Test
 For the presence of carbohydrate
moiety (cerebrosides and
gangliosides)
 Reagents: α- naphthol, 95%
EtOH, conc. H2SO4
 + result: violet ring at the
interface
 Principle: hydrolysis, dehydration,
condensation
3.) Test for N & P
 Reagents: fusion mixture (KNO3,
Na2CO3) – converts organic
compound into inorganic form
 Color after fusion: black to
grey/white
 N liberated as NH4 + H2O 
NH4OH (red litmus  blue
litmus)
 P H3PO4 + HNO3 + (NH4)3PO4
 (NH4)3 PO4•12 MoO4 ↓
(yellow crystalline)
4.) Kraut’s Test
 Detect choline (for lecithins and
sphingomyelin)
 Reagents: KI, bismuth subnitrate,
HNO3
 + result: dark orange or red
precipitate
 Principle: complexation reaction
5.) Ninhydrin Test
 Detects amino acid moiety
(lecithins, cephalins,
sphingomyelin)
 Reagents: triketohydrindene
hydrate
 + result: blue-violet solution
 Principle: oxidative
decarboxylation and deamination
followed by condensation
Thin-Layer Chromatography
 A type of adsorption chromatography
(mobile phase is either gas or liquid while
stationary phase is solid)
 Most widely used chromatographic
technique that uses adsorbents including
silica, alumina, florisil, and cellulose
 Mobile phase used: CHCl3-MeOH-NH4OH
(75:25:4)
 The Rf values for the various components
are (approximately):
 0.9 for phosphatidylethanolamine
 0.5 for phosphatidylcholine
 0.2 for sphingomyelin
 0.1 for lysophosphatidylcholine
 Sometimes the 3 lysophosphatidylcholine
cannot be detected but seems to appear as
a slight movement upward of the original
spot
 Two other components which are not
sufficiently well separated from
phosphatidylcholine are phosphatidylserine
and phosphatidylinositol. This makes the
phosphatidylcholine appear as a large
streaky spot.
Isolation of Lipids
  Complicated process due to
susceptibility of lipids to oxidation
and peroxidation
 Conditions that must be observed
during extraction:
1.) Tissues must be subdivided
under conditions that will not
bring about degradation of lipids
2.) Solvents to be use must
penetrate the tissue and break
protein-lipid bond
3.) Tissue must be washed
completely free of lipid by
repeated treatment with lipid-free
solvent

Extraction

Based on difference in solubility:

A. Triacylglycerols – extracted with

acetone or diethyl ether
B. Phosphoglycerides:
Lecithins – soluble in either, CHCl3,
C6H6, hot alcohol, CS2; insoluble in
acetone
Cephalins – same solubility as lecithins
but are insoluble in ethyl and methyl
alcohols
C. Glycolipids – insoluble in ether; more
soluble in acetone than phospholipids;
soluble in hot alcohol, CHCl3, C6H6
D. Cholesterol – soluble in ether, CHCl3,
C6H6, and hot alcohol and easily
crystallize from such solutions