Organic Molecules of Life - Exercise 2
Objectives
-Know the difference between a reducing sugar and a
non-reducing sugar.
-Distinguish Monosaccharides from Disaccharides and
Polysaccharides
-Understand the purpose of a control
-Understand what gives you positive and negative reactions
for the following assays and know what those assays are
testing for what specific type of sugar (mono, di, or
Polysaccharides): Benedict, Barfoed, Iodine, Biuret,
Miscibility, and Sudan Red. KNOW THE COLORS OF TUBES
WHEN THERE POSTIVE AND NEGATIVE FROM THE ASSAYS…
There are four major molecules, also known as
macromolecules, and these include carbohydrates,
proteins, lipids and nucleic acids. Each of these molecules
contain macromolecular varieties which can be chemically
degraded by hydrolysis (broken down into smaller pieces).
-Carbohydrates are polymers of monosaccharides
(monomer).
-Proteins are polymers of amino acids (monomer).
-Nuclei are polymers of nucleic acids (monomer).
-Lipids have the subcategories of neutral fats, and can be
digested into glycerol and fatty acids.
• Carbohydrates, proteins, and nucleic acids are
polymers (and they are made from smaller
monomers).
• Lipids are not polymers (they are not made from
monomers) You could say there are subunits in
lipids, such as, Glycerol & fatty acids.
Assay = Test
Carbohydrates – Complex Sugars
•
•
•
•
Monosaccharides – one sugar molecule
Disaccharides – two sugar molecules
Oligosaccharides – a few sugar molecules
Polysaccharides – many sugar molecules
What are Biological Molecules?


Carbohydrates

Proteins
 Sugar
 Amino acids
 Starch
 Collagen
 Cellulose
 Fibrin
Fats

Nucleic Acids
 Lipids
 DNA
 Fatty acids
 RNA
 Cell membranes
Carbohydrates
Monosaccharide which is the basic unit of a carbohydrate, and can’t be
broken down anymore (smaller molecules) or it can also be called simple
sugar. One significant group of Carbohydrates with 6 carbons is called
hexoses. The very common hexoses are glucose, galactose, and fructose.
Each of the three sugars have the exact same molecular formula, which is
C6H12O6, but there arranged differently, so these are called isomers. These
sugars also exist in different forms one is a ring and the other is a chain.
Monoasccharides in the chain form will have one carbon atom that is
doubled bonded to oxygen (C=O) which is known as a carbonyl group.
Solutions of these sugars will contain both the chain and ring
form in equilibrium, and each of the forms can freely convert in
to the alternative form under favorable chemical conditions.
Reducing Sugars
When talking about chemistry the main things to focus on are electrons, those are the
things that make reactions happen. You will learn more about this in organic chemistry.
An atom or molecules that receives one or more electrons form another substance is
said to have been reduced. The thing that donates an electron is known as the
reducing agent.
A reducing sugar is any sugar that possess a carbonyl group (C=O). Reducing sugars
are capable of donating an electron to another molecule. Reducing sugars can be
monosaccharides or disaccharides.
The chain form of any monosaccharide has the chemical group (C=O). In some, but not
all, disaccharides one of the two rings may be open, and they will have the C=O group
and thus be reducing sugars.
Those that cannot open one of the rings will not be a
reducing sugars, because it won’t be able to donate
its electrons.
All of these molecules are held by specific bonds, and these
bonds can be used to chemically identify the molecules that
posses them.
The test that you performed tested for the chemical
reactions of these bonds (simple chemical test) which are
also known as assays, and these test could can be used to
identify the presence of a particular type of molecules in a
simple mixture.
Reducing agent: Substance that loses electrons (oxidizes).
Oxidation agent: The electron lets the substance get oxidize.
Carbohydrates
Solutions of these sugars will contain both the chain and ring form in
equilibrium, and each of the forms can freely convert in to the
alternative form under favorable chemical conditions.
Monosaccharides
Glucose
Galtose
Fructose
Ribose
Monnose
Disaccharides
Polysaccharides
Sucrose
Starch
Maltose
Cellulose
Lactose
Glycogen
• What’s a control, what’s the
purpose of having it?
Control
• When doing an assay (test) a control test must be
done. The control tube will contain everything that
the assays contain except the substance to be
tested. In the assays, water is the solvent for all of
the other substance and will be in the control, along
with the test reagents.
• The control test is the negative standard against
which the assays are compared.
Page 22 – Lab Book
Page 22 – Lab Book
Monosaccharides: Glucose, Fructose, Galactose
Disaccharides: Lactose & Maltose
Polysaccharide: Starch
Sucrose Diasscahride: This always has rings, so it’s a non-reducing sugar.
You could put sucrose into any solution, and it’s the same thing as throwing
water, so nothing would change.
Benedict’s Assay for Reducing Sugars
The Benedict reagent is a clear blue solution of Copper
hydroxide (Cu (OH)2) at an alkaline pH (7). When this is
mixed with a solution containing a reducing sugar and
heater the Cu ion is reduced to cuprous form, which will
make the solution the solution would become turbid
(cloudy).
The color change in a positive assay can vary from greenish
to bright red, depending open the strength of the reducing
sugar (which determines how much precipitate will be
formed). A positive Benedict’s assay indicates the
presence of a reducing sugar either mono or
disaccharides.
Benedict’s Assay
Macromolecule
Monomer
Assay Name
Assay Indicates
Positive Result looks
like:
Carbohydrate
Monosaccharide
(simple sugar)
Benedict’s Assay
Presence of a
reducing sugar
(could be a
monosaccharide or
disaccharide)
A cloudy (turbid)
color change from
blue to greenish or
bight red color
Barfoed’s Assay for Monosaccharides
Monosaccharides can be distinguish from reducing disaccharides
by using Barfoed’s reagent, which is similar to Benedict’s, but set
to an acidic pH (about 4.5). In an acidic environment the
reducing disaccharides cannot reduce the cupric ion, but the
monosaccharide can.
All rings in disaccharide are closed; therefore, cant’ be reducing
sugar.
A positive Barfoed’s assay indicates the presence of a
monosaccharides and only monosaccharides, and is reenergize
by the production of a slight turbidity and a slight color change
from blue to red/violet or green. If you see particles in the
bottom of the test tube, it’s a positive result.
Barfoed’s Assay
Macromolecule
Monomer
Assay Name
Assay Indicates
Positive Result
looks like:
Carbohydrate
Monosaccharide
(simple sugar)
Barfoed’s Assay
Presence of a
monosaccharide
A slightly cloudy
(turbid) color change
from blue to green or
red/violet (looks for
redish particles at
bottom of tube)
Summary of Benedcit’s & Barfoed’s Assay
Note: Those that cannot open one of the rings is not a reducing sugar.
• Benedcit’s assay: Used to test for monosaccharides and disaccharides.
It has a pH of about 7, but when the solution gains (reduces) an electron
from copper ion the solution becomes a reducing sugar (positive result).
In the Benedcit’s assay five carbohydrates were reduced - three were
monosaccharides, and the other two were disaccharides.
• Barfoed’s Assay was used for testing monosaccharides too.
Monosaccharides can be distinguish from reducing disaccharides by
using this test. But Barfoed’s test had a pH of 4.5, which is a more acidic
environment. A positive result indicates presence of monosaccharides
and only monosaccharides.
• In an acidic environment, disaccharides can’t gain an electron from the
copper ion, but monosaccharides can gain an electron from copper
ion. Those that cannot open one of the rings will not be a reducing
sugars, because it won’t be able to donate its electrons.
Starch: Iodine Assay
Starch is a polysaccharide composed of hundreds of units of
glucose covalently linked together. The structure of starch is a
such that the molecule is coiled. Iodine [I2KI] will interact
specifically with these coiled molecules to form a blue to black
colored complex.
Note: Iodine will not react with other carbohydrates, thus
retaining its original color of yellow or amber. If it is still the
color of yellow or amber but diluted somewhat, the assay is
negative, but if it is any other color, such as, brown to reddish
brown to blue to black, then its positive.
A positive iodine assay indicates the presence of starch, so if the
color turns dark blue, or black, this is a positive result.
The Iodine Assay - Starch
Macromolecule
Monomer
Assay Name
Assay Indicates
Positive Result
looks like:
Carbohydrate
Monosaccharide
The Iodine Assay
Presence of
Starch
A color change
from yellow to
reddish brown or
bluish/black
Proteins (peptide bonds) - Biuret Assay
Proteins are composed of atoms or elements C,H,O, N, & S. Proteins are
very large polymers. The monomer unit of proteins is an amino Acid.
Although each different protein has a unique sequence of amino acids,
the covalent bonds between adjacent amino acids are essentially the
same.
A covalent bond between two amino acids is called a peptide bond.
The presence of peptide bones can be determine by using Biuret test.
In a strongly alkaline environment (pH 8-14), copper sulfate reacts with
the peptide bones, causing the blue color of the reagent to change to a
violet color.
A violet color indicates a positive Biuret assay and also indicates the
presence of peptide bonds and of course of proteins.
Polypeptides
• Polypeptides
– Are polymers of amino acids
– A peptide bond is a covalent bond between 2
amino acids.
• A protein
– Consists of one or more polypeptides
Page 24 – Lab Book
Page 24 – Lab Book
Biuret Assay- Protein
Macromolecule
Monomer
Assay Name
Assay Indicates
Positive Result
looks like:
Protein
Amino acids
Biuret Test
Presence of
peptide bonds
Color change
from blue to
violet
Lipid - Miscibility
All lipids are insoluble in water. Families of lipids, including fats and oils,
phosopholipids, steroids, and carotenoids. We will look at triglycerides in lab.
Fats and oils are polymers. The subunit of fats/oils are glycerol and three fatty
acids.
Miscibility Assay: If a fat or oil mixes with water in the test tube, two distinct
layers of liquid will form because oil and water do not mix, this would
indicate a negative result (two layers of liquid), but if two the substances
mix, then it’s a positive result. Basically what this means is that the substance
assayed does not mix with water (negative miscibility).
“Friendship and money…oil and water.”
-Michael Corleone (Godfather III)
Charged polar end
Lipids: Fatty Acids

Long hydrocarbon chains



Very hydrophobic
Oil and water don’t mix
Used for energy storage

Very high energy

Long-term storage
Lipid tail end
• What’s so important about
lipids, what barrier do they
create in a cell?
• Hint it has multiple heads &
tails.
Phospholipid bilayer - Membrane Structure
Fluid Mosaic Model: fluid membrane with various proteins embedded in or attached
to a double layer (bilayer) of phospholipids.
WATER
Hydrophilic
head
WATER
Hydrophobic
tail
Page 25 – Lab Book
Page 25 – Lab Book
Miscibility Assay - Lipid
Macromolecule
Subunit
Assay Name
Assay Indicates
Positive Result
looks like:
Lipid
Glycerol + fatty
acid
Miscibility Assay
Presence of an
insoluble
(hydrophobic)
substance
Here we look for a
negative result,
namely no mixing. If
an insoluble substance
is present, there will
be two visible layers.
Lipid - Sudan Red Assay
Sudan Red Assay: The Sudan stain will mix
with fats and oils and stain them but will not
stain water and water soluble compounds.
A positive Sudan red assay indicates the
presence of a lipid such as fat or oil and vice
versa. So if the substance stains red, the
assay is positive.
Page 25 – Lab Book
Page 25 – Lab Book
Sudan (Red) Assay - Lipid
Macromolecule
Subunit
Assay Name
Assay Indicates
Positive Result
looks like:
Lipid
Glycerol + fatty acid
Sudan (Red)
Assay
Presence of a
lipid
Lipids (but
water) will stain
Questions
• 1. You are given two unlabeled containers and told that one
of them contains a solution of pure glucose and the other
contains a solution of pure maltose, a disaccharide. How can
you identify the content of the bottle by performing chemical
assays?
• 2. What is a monosaccharide? Why all monosaccharides and
most disaccharides are reducing sugars?
• 3. In solution, chain and ring forms of glucose molecules exist
in equilibrium. Explain this statement?
• 4. Both fructose and glucose are reducing sugars. When
these two simple sugars become chemically bonded, a
disaccharide, sucrose, is formed. Why sucrose is not a
reducing disaccharide?
Questions
•
1. You are given two unlabeled containers and told that one of them contains a solution of
pure glucose and the other contains a solution of pure maltose, a disaccharide. How can
you identify the content of the bottle by performing chemical assays? You can perform the
Bendicts and Barfoed’s assay to see what reactions are positive. Maltose will only be
positive in the Bendcit’s assay due to the pH (7) environment. And, if you perform the
Barfoed’s assay and get a negative result, then you know you got Benedict’s assay for
maltose. For glucose you get a positive reaction for the Bendict’s assay and a negative
result for Barfoed’s assay.
•
2. What is a monosaccharide? Why all monosaccharides and most disaccharides are
reducing sugars? A monosaccharide is a simple sugar that contain a carbonyl group. They
can accept (gain) the Cu ion under acceptable pH levels, for instance, pH 7.
•
3. In solution, chain and ring forms of glucose molecules exist in equilibrium. Explain this
statement? Solutions of these sugars will contain both the chain and ring form in
equilibrium, and each of the forms can freely convert in to the alternative form under
favorable chemical conditions.
•
4. Both fructose and glucose are reducing sugars. When these two simple sugars become
chemically bonded, a disaccharide, sucrose, is formed. Why sucrose is not a reducing
disaccharide? Sucrose always has rings, so it’s a non-reducing sugar. You could put
sucrose into any solution, and it’s the same thing as throwing water, so nothing would
change.
Questions
• 5. Write three names of monosaccharides.
• 6. Write two names of reducing disaccharides.
• 7. What is the difference between reducing
disaccharide and non-reducing disaccharides?
• 8. What are three names of polysaccharides.
Questions
• 5. Write three names of monosaccharides.
Glucose, Fructose, & Galactose
• 6. Write two names of reducing disaccharides.
Lactose & Maltose
• 7. What is the difference between reducing disaccharide and
non-reducing disaccharides? The environment, in our case,
the pH was the factor. Under alkaline conditions a
disaccharide could gain an electron from the copper ion,
but in an acidic environment (pH 4.5) the disaccharide
can’t accept the electron from the copper ion.
• 8. What are three names of polysaccharides.
Starch, Glycogen, & Cellulose
a.
b.
c.
d.
e.
Sucrose
Maltose
Starch
Glucose
Egg albumin
Questions
• 9. This substance gives a negative Benedict’s assay, a negative Barfoed’s
assay, a positive iodine assay and a negative Biuret assay.
• 10. This substance gives a positive Benedict’s assay, a positive Barfoed’s
assay, a negative iodine assay, and a negative Biuret assay.
• 11. This substance gives a positive Benedict’s assay, a negative Barfoed’s
assay, a negative iodine assay, and a negative Biuret assay.
• 12. This substance is positive in Biuret assay and negative in all other
assays.
a.
b.
c.
d.
e.
Sucrose
Maltose
Starch
Glucose
Egg albumin
Questions
• 9. This substance gives a negative Benedict’s assay, a negative Barfoed’s
assay, a positive iodine assay and a negative Biuret assay. C - Starch
• 10. This substance gives a positive Benedict’s assay, a positive Barfoed’s
assay, a negative iodine assay, and a negative Biuret assay. D - Glucose
• 11. This substance gives a positive Benedict’s assay, a negative Barfoed’s
assay, a negative iodine assay, and a negative Biuret assay. B - Maltose
• 12. This substance is positive in Biuret assay and negative in all other
assays. E - Egg albumin
Questions
a.
b.
c.
d.
e.
Sucrose
Maltose
Starch
Glucose
Egg albumin
• 13. All test (Bendict’s, Barfoed’s, iodine, and Biuret) on the substance came
negative.
============================================================
• 14. Benedict’s assay is for ______________ Positive reaction for Benedict’s
assay produces _________ color.
• 15. Barfoed’s assay distinguishes between _________ and
_______________.
• 16. A protein solution can be identified by performing the _________assay.
Questions
a.
b.
c.
d.
e.
Sucrose
Maltose
Starch
Glucose
Egg albumin
• 13. All test (Bendict’s, Barfoed’s, iodine, and Biuret) on the substance came
negative. A - Sucrose
============================================================
• 14. Benedict’s assay is for reducing sugars Positive reaction for Benedict’s
assay produces red color.
• 15. Barfoed’s assay distinguishes between monosaccharide and reducing
sugar.
• 16. A protein solution can be identified by performing the Biuret assay.