Analysis of starch, sugar, protein, lipid and

amino acids
1. Monosaccharide
2. Disaccharides
3. Polysaccharides
4. Carbohydrate Detection Method
 Review on Carbohydrate
• most abundant class of organic compounds
• originate as products of photosynthesis
n CO2 + n H2O + energy  CnH2nOn + n O2
• classified according to the number of simple sugar units

What are the functions?

• Store energy in the form of starch (in plants) or glycogen (in animals &
humans).

• Supply carbon for synthesis of other compounds.

• Form structural components in cells and tissues

 CLASSIFICATION OF DIETARY CARBOHYDRATES
Monosaccharides Glucose, fructose, galactose
Disaccharides Sucrose, lactose, maltose
Polysaccharides Starch: Amylose, amylopectin
Non-starch: Cellulose, pectins
Classes of carbohydrate

1. monosaccharide

• simplest sugar
•Glucose  "blood sugar"
• Fructose  "fruit sugar"
• Galactose the breakdown of lactose.

glucose

D-glucose
(ring structure)
 Classes of carbohydrate

2. disaccharide

• made by connecting two simpler molecules.

 Classes of carbohydrate
3. polysaccharide • e.g. Starch, Cellulose, Chitin, Glycogen

a. Starch : 2 types

1. Amylose 2. Amylopectin
- unbranched - highly branched
- quite soluble in water, but - water insoluble and represents
only represents 20% of the the other 80% of the starch.
Starch.
 Classes of carbohydrate

b. Cellulose

• fn: structural component

 Classes of carbohydrate

c. glycogen

• linkage similar to amilopectin BUT the branching is more numerous

• Our bodies make glycogen from the unused carbohydrate

glycogen
 Classes of carbohydrate

d. chitin

• Similar to cellulose in structure

and function
• BUT the unit = N-acetyl-ß-D-
glucosamine
• chitin has an amide group
instead of a hydroxyl group
(alcohol)

cellulos c
e
 Molish
 Barfoed
 Bennedict
 Iodin test
 Detect all carbohydrates
 Principle: dehydration of the carbohydrate by
sulphuric acid to produce an aldehyde, then
condenses with the phenolic structure
forming a purple ring.
 Shows positive test for: Monosaccharides

 Reactions: Reducing monosaccharides are oxidized by the copper ion in

solution to form a carboxylic acid and a reddish precipitate of copper (I)
oxide within three minutes.

 Use Barfoed's reagent (a solution of cupric acetate and acetic acid).

 The formation of a reddish precipitate within three minutes.

 Detect reducing sugars (monosaccharide’s and some disaccharides),
which have free ketone or aldehyde functional groups.

 Reaction of reducing sugars and Benedicts reagent causes the Benedicts

reagent to change color. The color varies from green to dark red (brick)
or rusty-brown, depending on the amount of and type of sugar.
 Detect aldose sugars (sugar with aldehyde
groups).
 Detect polysaccharide eg
starch.

 Starch turns into an

intense "blue-black"
colour upon addition of
triiodide anion solution,
due to the formation of an
intermolecular charge-
transfer complex.
1. Amino acids
2. Protein structure and function
3. Enzymes
4. Protein quantification techniques
•Amino acids are organic molecule with at least one carboxyl
group and at least one amino group.
•Several hundreds amino acids are known to present but 20 of
them are usually found in proteins
• The side chain (R) determine the identity of particular amino acid:
- non-polar
- polar
- acidic
- basic
 Amino acids can be linked by
forming bond between α-carboxyl
group of one amino acid and the α-
amino group of the next one
 Peptides are compounds formed by
linking small numbers of amino
acids
 When many amino acids are linked
by peptide bonds, they form a
polypeptide chain
 Proteins are polypeptides with more
than 100 amino acids
 Protein can also be hydrolyse (break
down) into smaller peptides and free
amino acids.
 The chemical reaction is called
hydrolysis by acid-, base- or
enzyme catalysts.
Other biological function of amino acids:
 Branched-chain amino acid (isoleucine, leucine, valine): cannot
be synthesized but essential to human body in certain condition
 Monosodium glutamate (MSG) is the derivative of glutamic acid
that serve as flavour enhancer but causes physiological reaction
 Serotonin is the derivative of tryptophan that plays a key role in
sudden infant death syndrome (SIDS)
 Histamine is histidine with the acid group removed which
involve in immune response
Size of protein is defined using dalton unit

Protein composition and behaviour:

1) Monomeric (single polypeptide chain) vs oligomeric (consist of

2 or > polypeptide chain)
2) Simple protein (consist of only amino acid residues) vs
conjugated protein (contains amino acids and other chemical
group/ metal atom)
3) Globular protein (water soluble) vs fibrous protein (water
insoluble)
 Globular protein that serve as catalyst
1. Increase rate of reaction by lowering the
activation energy barrier.
2. It is not used up or permanently changed
during the catalytic process.
3. It does not change the position of
equilibrium only the rate at which
equilibrium is attained.
4. It usually acts by forming a transient
complex with the reactant, thus stabilizing
the transition state.
 Spectroscopic procedures

 Measurement of the total protein content by

colorimetry (eg. Spectrophotometer)

 Amino acid analysis (eg. Mass spectrometry/

protein sequencing)

 Other methods: radiolabelling of proteins,

HPLC etc
 Based on polypeptide
chelation of cupric ion in
strong alkali

 Compounds containing 2
or more peptide bonds
react with cupric ions
(Cu2+) in alkaline solution
to produce a complex of
reddish-purple colour
 Used to measure the concentration of
substances in solution
 Used optical instrument known as
spectrophotometer (Absorbance)
 Protein’s presence is measure based on the
color change (light intensity being absorb)
 a heterogeneous group of compounds,
including fats, oils, steroids, waxes, some
vitamins
 a compound that is insoluble in water, but
soluble in an organic solvent (e.g., ether,
benzene, acetone, chloroform)
 important in biological systems because they
form the cell membrane, a mechanical barrier
that divides a cell from the external
environment.
 form of stored energy in biological systems
 several essential vitamins are lipids.
 serves as a thermal insulator in the
subcutaneous tissues and around certain
organs.
1. Simple lipids: Esters of fatty acids with
various alcohols.
◦ Fats
◦ Waxes
2. Complex lipids: Esters of fatty acids
containing groups in addition to an alcohol
and a fat
◦ Phospholipids
◦ Glycolipids
◦ Other complex lipids
3. Precursor and derived lipids:
 A fatty acid is a molecule characterized by the
presence of a carboxyl group attached to a
long hydrocarbon chain.
◦ Saturated fatty acids
◦ Unsaturated fatty acids

General structure of fatty acid

1. In a cis stereoisomer, two similar groups
attached to the carbon double bond are
found on the same side.
2. In a trans stereoisomer, two similar groups
attached to the carbon double bond are
found on opposite sides.
 is a fatty acid triester of glycerol.
 Glycerol has three hydroxyl groups.
 Fatty acids can be attached at these three
sites forming a triglyceride.
◦ Short-chain unsaturated triglycerides are liquid at
room temperature.
◦ Long-chain saturated triglycerides are solid at room
temperature.
 composed of two fatty
acids, a glycerol unit, a
phosphate group and a
polar molecule.
 The phosphate group
and polar head region of
the molecule is
hydrophillic (attracted to
water), while the fatty
acid tail is hydrophobic
(repelled by water).
 Known as sterol -combination of a steroid
and an alcohol.
 It is an important component of cell
membranes and is also the basis for the
synthesis of other steroids.
 In the cell membrane, the steroid ring
structure of cholesterol provides a rigid
hydrophobic structure that helps boost the
rigidity of the cell membrane.

Cholesterol
structure
Qualitative analysis Quantitative analysis

Solubility test Chromatoghraphy analysis

Emulsion formation Saponification Value

Libermann Burchard Test Iodine value

Sudan Stain Test Direct mass calculation

 To test the solubility of oils in different
solvent.
 Fats are not dissolved in water due to
their nature: non-polar (hydrophobic),
but soluble in organic solvents (eg.
choloroform).
 Different lipids have ability to
dissolve in different organic
solvent-enable to separate
a mixture of fat from
each other.
 To test the presence of broad group of lipids.
 Lipids are non-polar organic compounds,
hence they are soluble in organic solvents such
as ethanol but insoluble in water.
 Ethanol extracts the lipid.
 The lipid spontaneously comes out of solution
when water is added and is dispersed as
micelles (small droplets) throughout the
solution of ethanol and water.
 Showing a layer of cloudy white
suspension forms at the top of the solution.
 The Liebermann–Burchard or acetic anhydride
test is used for the detection of cholesterol.
 The formation of a green or green-blue color
after a few minutes is positive.
 Lieberman–Burchard is a reagent used in
a calorimetric test to detect cholesterol,
which gives a deep green color.
 One of the most powerful analytical
procedures for separating and analyzing the
properties of lipids.
 can be used to identify the chemical structure
of the peaks, e.g., mass spectrometry.
 Various forms of chromatography are
available to analyze the lipids in foods:
◦ thin layer chromatography (TLC),
◦ gas chromatography (GC),
◦ high pressure liquid chromatography (HPLC)
 The saponification number is a measure of
the average molecular weight of
the triacylglycerols in a sample.
 Saponification is the process of breaking
down a neutral fat into glycerol and fatty
acids by treatment with alkali.
 The saponification number is defined as the
mg of alkali required to saponify one gram of
fat.
 The iodine value (IV) gives a measure of the
average degree of unsaturation of a lipid
 The higher the iodine value, the greater the
number of C=C double bonds.
 By definition the iodine value is expressed as
the grams of iodine absorbed per 100g of
lipid.