BIOCHEMISTRY is ❤️

EXPERIMENT 5 – LIPIDS (Fats)

 Lipids are a large and diverse group of natural occurring organic compounds that are
related by their solubility in non-polar organic solvents.

“Non polar solvents contain bonds between atoms with similar electronegativities, such
as carbon and hydrogen. Bonds between atoms with similar electronegativities will lack
partial charges; it’s this absence of charge which makes these molecules “non-polar”.
(e.g Ether, Chloroform, Acetone, & Benzene)

 The common feature of these lipids is that they are all esters of moderate to long chain
fatty acids.

“an ester − as opposed to an ether − is a chemical compound derived from an acid

(organic or inorganic) in which at least one –OH (hydroxyl) group is replaced by an –O–
alkyl (alkoxy) group. Usually, esters are derived from a carboxylic acid and an alcohol.”

 These long-chain carboxylic acids are generally referred to by their common names,
which in most cases reflect their sources. Natural fatty acids may be saturated (sol.)or
unsaturated (liq.).

 Saturated acids have higher melting points than unsaturated acids of corresponding
size.

Fatty acids normally found in foods vary in:

- Chain Length
- Degree of Unsaturation
- Position on the Glycerol molecule.

Elements present in Lipids

 - Carbon
- Hydrogen
- Oxygen
Combined as esters of various fatty acids with
- Tri-hydroxy alcohol
- Glycerol

 Saponification is the process in which lipid molecule is hydrolized with an alkali such as
NaOH (Sodium Hydroxide, a strong base), splits into glycerol and fatty acids.
 Soap is sodium salt of the fatty acid. The product of saponification.

 The cleansing power of soap and detergent is due to their emulsifying action and their
ability to reduce surface tension.

“An emulsifier or emulsifying agent is a compound or substance that acts as a stabilizer

for emulsions, preventing liquids that ordinarily don't mix from separating.”

 The soap molecule contains polar head (which interacts with the (-) negative portion of
the aqeous phase), and a non-polar tail (which dissolves in the oil droplets.)

 Fats develop rancid odor and taste when exposed to air at room temperature.
Peroxides, volatile aldehydes, ketones and acids, are responsible for the rancid odor
because of the combination of double bonds of unsaturated fatty acids with oxygen.

 Fatty acid residues may differ in chain length and be distinguished by the difference in
intensity of color due to the absorption of Sudan III.

“Sudan III is a red fat-soluble dye that is utilized in the identification of the presence of
lipids, triglycerides and lipoproteins. The Reaction: Sudan III reacts with the lipids or
triglycerides to stain red in color. This test is conducted to test for the presence of lipids
in a solution.”

 The fatty acid residues may also differ according to the extent of saturation (number of
double bonds or unsaturated) present in hydrocarbon chain. It is demonstrated by the

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 degree of decolorization of halogen solution (iodine + bromine) that is usually measured
by the iodine number.
 The more double bonds a fat contains, the more iodine required for the addition
reaction; thus, a high iodine number means a high degree of unsaturation.

 Solubility Test is the preliminary test which detects the presence of all lipids. This test
detects the solubility of lipid in various solvents to check whether it is miscible or
immiscible in polar or non-polar solvents.

Lipids are readily miscible in non-polar solvents like chloroform, ether, acetone
&benzene,partially soluble in a polar solvent like ethanol and immiscible in a polar
solvent like water.

 Acrolein Test is the standard test for fats. It is when a fat is heated strongly in the
presence of a dehydrating agent such as KHSO4 (Potassium Sulfate), the glycerol portion
of the molecule is dehydrated to form acrolein, the unsaturated aldehyde which has the
peculiar odor of burnt grease.

Positive result: If glycerol present in the sample it will give a pungent smell.

Negative result: If glycerol is absent in a sample, then it will not produce a pungent smell.

 Test for Unsaturation of Fatty Acids is used to detect the presence of unsaturated fatty
acids or the amount of double bond in a lipid sample.

“All the neutral fat contains glycerides of fatty acids. Double bond found in the structure
of unsaturated fatty acids which becomes saturated by taking up either bromine or
iodine. If the lipid contains more unsaturated fatty acids or more double bonds that
means, it will take more iodine.”
The number of drops determines the taking up of iodine by the unsaturated fatty acid of
lipids.

Positive result: Pink colour/ faint orange color will disappear by the addition of
unsaturated fatty acids.

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 Negative result: Pink colour/faint orange color will not disappear.

 Test for Randicity / Free Fatty Acids This is based on the “Neutralization reaction”
where the alkali is neutralized by the addition of free fatty acids in the lipids.

 Lieberman-Burchard Test was first given by the scientist “Liebmann” to detect the
presence of cholesterol.

Cholesterol reacts with the strong concentrated acid i.e. sulphuric acid and acetic
anhydride. Sulphuric acid and acetic anhydride act as a dehydrating and oxidizing agent.

Positive result: It indicates the presence of cholesterol in a sample by giving bluish-green

colour to the solution.

Negative result: It is indicated by no colour change.

 Saponification TestIt is based on the “Saponification reaction”, where the triglycerides

of lipid react with an alkali NaOH and produce soap and glycerol in the presence of
ethanol. This reaction also refers to as “Alkaline hydrolysis of esters”.

 Emulsification test of Lecithin is used to detect the presence of lipids

“Emulsification is the process which stabilizes the water and oil emulsion, by the help of
emulsifying agents. The lipid or oil in water appears on the top of the water because of
the high surface tension of water which gets together to form a separate layer. On the
addition of emulsifying agents like bile salts, soap etc.”

“Emulsifying agents emulsify the lipid by which the lipid appears as the tiny droplets
suspended in the solution.”

EXPERIMENT 6 – Proteins

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  Proteins consist of amino acids which are characterized by the -CH(NH2)COOH
substructure. It is also a classification of peptides consisting of 50 or more amino acids.

 Amino acids is the combination of amino group (nitrogen + 2 hydrogen = -NH2) and acid
entity is the carboxylic group (-COOH). It is the basic building blocks of enzymes,
hormones, proteins and body tissues.

 Peptide bond -C(=O)NH- is the link between amino acids when the carboxyl group of
one molecule reacts with the amino group of another molecule. Hence, releasing water
molecule (H2O).

 Peptide is a compound consisting of 2 or more amino acids.

 Oligopeptides have 10 or fewer amino acids.

 Polypeptides are chains of 10 or more amino acids.

 Biuret Test is used for the quantitative photonetrical determination of total protein
concentration. The intensity of the color produced in this test is proportional to the
number of peptide bonds participating in the reaction.

Positive result: Peptides and proteins (long chain polypeptides) react with Cu2+ in
alkalinity to create a positive result, blue violet colored complex.

Amino acids and copper (II) ions form a blue complex.

 Xanthoproteic Test is when the nitro derivatives (nitrated aromatic rings of amino acids
like tyrosine and tryophan with the use of 65% nitric acid) show an intensely yellow
color. It is because nearly all proteins contain aromatics, taken as a protein-test either.

 Millon’s Test (Millon’s reagent) an analytical reagent used to detect soluble proteins. It
consists of mercury dissolved in nitric acid. Millon’s test is not specific for proteins, it
actually detects phenolic group which are present in amino acid tyrosine.

Positive result: a reddish-brown coloration or precipitate indicates the presence of

tyrosine residue which occur in nearly all proteins.

 Sulfur Test a process where sulfur-containing amino acids are boiled with an alkali, the
sulfhydryl or disulfide groups are converted to an inorganic sulfide, Na2S. This reacts

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 with lead acetate to form a black precipitate of PbS. Cysteine and cystine in the free
state and in proteins give a positive result to the sulfur test.

 Hopkins-Cole Test it is where indole ring of typtophan condenses with glyoxalic acid in
the presence of sulfuric acid to form violet-colored complex.

 Ninhydrin Test is a test for amino acids and proteins with a free -NH2 group. When such
an amino group reacts with ninhydrin, a purple-blue complex is formed.

Experiment 7 – Denaturation of Proteins

 Denaturation of proteins involves the disruption and possible destruction of both the
secondary and tertiary structures. Since denaturation reactions are not strong enough
to break the peptide bonds, the primary structure (sequence of amino acids) remains
the same after a denaturation process.

Denaturationdisrupts the normal alpha-helix and beta sheets in a protein and uncoils it
into a random shape.

Tertiary Structure: Four types of bonding b/w “side chains”

- Hydrogen bonds
- Salt Bridges
- Disulfide bonds
- Non polar hydrophobic interactions

 Precipitation or Coagulation is the most common observation in the denaturation

process.

 Alcohol disrupts hydrogen bonding

 Hydrogen bonding occurs between amide groups in the secondary protein structure. It
also occurs between “side chains” in tertiary protein structure in a variety of amino acid
combinations.

 70% alcohol sol’n is used as a disinfectant on the skin. This concentration of alcohol is
able to penetrate the bacterial cell wall and denature the proteins and enzymes inside
the cell.

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  95% alcohol sol’n merely coagulates the protein on the outside of the cell wall and
prevents any alcohol from entering the cell.

 Alcohol denatures proteins by disrupting the side chain intramolecular hydrogen

bonding.

 Heat can be used to disrupt hydrogen bonds and non polar hydrophobic interactions.
This occurs because heat increases the kinetic energy and cause the molecules to
vibrate so rapidly and violently that the bonds are disrupted.

 Alkaloidal reagents (e.g tannate & trichloroacetate) are high molecular weight anions.
The negative charge of these anions counteracts the positive charge of the amino group
in proteins giving a precipitate.

 Inorganic acids disrupt salt bridges held together by ionic charges.

 Salt bridges result from the neutralization of an acid and amine on side chains. The final
interaction is ionic between the positive ammonium group and negative acid group.

 Double replacement reaction occurs where the positive and negative ions in the
saltchange partners with the positive and negative ions in the new acid. This reaction
occurs in the digestive system, when acidic gastric juices cause curdling or coagulation
of milk.

 Heavy metal salts act to denature proteins in much the same manner as inorganic acids.
It may also disrupt disulfide bonds because of their hight affinity and attraction for
sulfur and will also lead to the denaturation of proteins.

The reaction of a heavy metal salt with a protein usually leads to an insoluble metal
protein salt.

Heavy metal salts contains:

- Hg+2
- Pb+2
- Ag+1
- TI+1
- Cd+2

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  Heavy Metal Ions precipitate proteins from their solutions by cross-linking free amino
groups and carboxylate groups.

 CuSO4 - Copper Sulfate

 NaOH - Sodium Hydroxide (A strong base, stronger than Ba(OH)2)
 Ba(OH)2 - Barium Hydroxide (A slightly strong base, stronger than NH4OH)
 NH4OH - Ammonium Hydroxide (A weak base)
 HCl - Hydrogen Chloride or Hydrochloric Acid (A very strong acid, stronger than H2SO4)
 H2SO4 - Hydrogen Sulfate or Sulfuric Acid (A strong acid, stronger than HNO3)
 HNO3 - Hydrogen Nitrate or Nitric Acid (An acid stronger than acetic acid)
 Pb(C2H3O2)2 - Lead Acetate also called Lead Sugar or Salt of Saturn
 HgCl2 - Mercury Chloride or Mercuric Chloride
 Cd(NO3)2 - Cadmium Nitrate
 KHSO4 - Potassium Sulfate (they are acidic salts that are soluble in water. They also
react as acids to neutralize bases.)
 CCl4 - Carbon Tetrachloride
 CHCl3 - Chloroform or Trichloromethane (a central nervous system depressant which
can also cause respiratory and cardiac failure with sufficient exposure.)
 Alcoholic KOH - Ethanolic Potassium Hydroxide (a solution of Potassium Hydroxide and
Ethanol, which is an alcohol)
 CaCl2 - Calcium Dichloride
 MgCl2 - Magnesium Chloride