LAB NO: 5

Carbohydrates (Saccharides)
 Saccharides or also called as carbohydrates are sugars that alongside fat and
protein, theyare one of the three macronutrients to provide energy, the main
source of energy.

 Catalytic effect of chlorophyll in plants yields a group of substances that are called
“glucides”. Previously, these substances were named under the general name
“carbohydrates” for the formula (CH2O)n; such as formaldehyde (CH2O)1, acetic
acid (CH2O)2, lactic acid (CH2O)3, etc. Later studies have shown that such
substances contain the same chemical formula but possess different properties. In
terms of their structure; rhamnose, mannitol, and digitalose have to be
considered as carbohydrates. But these three substances do not fit into the group
of carbohydrates because of their chemical formula. In this manner, new term
(glucide) and nomenclature has been found for this kind of molecules. However,
the term carbohydrate is still in use.

 Sugar is widely present in the plant kingdom and it is a major component of plant
material which plays an important role in photosynthesis of green plants, a major
source of energy needed to maintain the life of animals and plants.

 According to the structure and degree of polymerization saccharides can be divided

into monosaccharides, disaccharides and polysaccharides.

*Polymerization: Small molecules called as monomers, combine chemically to

produce largechainlike molecule called polymer.

 The bond between the glucide molecules (the combining bond) can be degraded
through boiling/heating in a solution with diluted mineral acids or some fermentation
agents under certain (appropriate) conditions into its building blocks. Thus, these
molecules can be processed through hydrolysation into its simplest molecules.

 They are organic compounds composed of three elements; carbon, hydrogen, oxygen, at
least 2 hydroxyl (OH) and 1 reducing unit (can be either aldehyde or ketone).

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  Monosaccharides are simplest form of carbohydrates. Composed of –C, -H, -O, -OH and
reduction unit. The number of carbon atoms in the monosaccharide is no more than 10.
Naturally occurring and biologically important monosaccharides are 5 to 6 carbon atoms.
Monosaccharides are water soluble, crystalline solids.

 To distinguish between these simple molecules, they are usually named based
upon their structure (general chemical naming of a molecule). Thus, if reduction
unit of a monosaccharide is an aldehyde, this molecule’s basic name is aldose,
such as glucose. But if reduction unit is a ketone, its general name is ketose, such
as fructose.

*BUT* Ketoses must first tautomerize to aldoses before they can act as reducing sugars.
If reduction unit is is aldehyde called as aldose sugar, if reduction unit is ketone called as ketose
sugar.

Tautomerization: Functional isomerism, migration of atom, generally H.

 Another rule of naming a monosaccharide depends on the number of “O” atoms in a

molecule which are in hydroxyl (-OH) groups or in reduction unit. But, nowadays the
naming of a molecule through an “O” (oxygen) atom is replaced by a “C” (carbon) atom,
where it has the same logic. Here, instead of oxygen atom(s) the number of carbon
atom(s) is considered. Thus, biose (molecule containing 2 oxygen atoms or 2 carbon
atoms), triose (3O or 3C), tetrose, pentose (e.g. rhamnose, arabinose), hexose (e.g.
glucose), heptose are the general names used based upon the number of oxygen or
carbon atoms.

 Disaccharide is a combination of two monosaccharide. Eg. sucrose, maltose, and lactose

 The polysaccharide is made up of hundreds or even thousands of glucose molecules. Eg.

starch, liver glycogen and muscle glycogen

**Sugars are divided into 2 groups: a) Reducing (Hemiacetal/Hemiketal)

b) Non-reducing (Ketal/Acetal)

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 How to Identify Reducing and Non-Reducing Sugars?
You can identify a sugar by looking for the anomeric carbon (C bound to 2 oxygen atoms, See figure
4). If that anomeric carbon has an OH group coming (hemiacetal) off then it is reducing sugar.

The sugar in sweets, candies etc. have OH groups oriented in the perfect way to interact with
receptors on our tongue, which creates the sweet taste!

Part of the orientation of these OH groups is a hemiacetal. In fact, the sugar glucose may be the
most commonly known hemiacetal. A hemiacetal is a carbon connected to two oxygen atoms,
where one oxygen is an alcohol (OH) and the other is an ether (OR).**

Non-reducing sugars

Non-reducing sugars do not have an OH (hemiacetal) group instead they have acetals (ketals)
attached to the anomeric carbon so they cannot reduce other compounds. Generally disaccharides
(besides few exception) and polysaccharides are non-reducing sugars.

Figure 1: Formation of Hemiacetals, Hemiketals, Acetals and Ketals

*Hemiacetal/Acetal → Aldose Sugars

*Hemiketal/Ketal → Ketose Sugars

*Hemiacetals/Hemiketals are in equilibrium with Aldehyde/Ketone

*Acetals/Ketals are not in equilibrium with Hemiacetal/Hemiketals

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 Figure 2: Sucrose as non-reducing sugar

Not all disccaharides are non-reducing sugars, some have reducing property!

Most of the di-, oligo-, and polysaccharides have two distinct ends. The end that has a hemiacetal
(orhemiketal) on its terminal is called the reducing end, and the one that does not contain a hemiacetal
(or hemiketal) terminal is the non-reducing end.

Non-reducing end Reducing end

Figure 3: Lactose as reducing sugar

Polysaccharides, such as amylose or amylopectin, do have a hemiacetal group on one of their

terminal ends, but they are mainly nonreducing substances because there is only one reducing group
present for every 2,000–10,000 monosaccharidic units. In such a low concentration, the reducing
group does NOT give a positive test with Benedict’s or Fehling’s reagent.

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 2 Ethers

1 Alcohol and 1 Ether

Anomeric Carbon

Figure 4: Identify reducing – non reducing sugars by looking at the hemiacetal / acetal / anomeric
carbon.

* All monosaccharides are reducing sugars.

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Figure 5: Hemiacetals

Figure 6: Fischer Projections of Glucose

Figure 7: Ring Chain Tautomerization of Glucose “Pyranose” Form

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Figure 8: Ring Chain Tautomerization of Glucose “Furanose” Form

We’ve seen five separate isomers so far: the straight chain form, the pyranose form (alpha and beta),
and the furanose form (alpha and beta).

In aqueous solution, these five forms are all in equilibrium with each other!

When you dissolve glucose in water, here’s the distribution you get:

Figure 9: Isomers of Glucose in Water

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 Lab No 5: Qualitative Determination of Oses (Monosaccharide)
Plant Sample: Camellia sinensis

Determination of Oses (Monosaccharides):

Classical Determination Methods

a. Reduction of Metal Salts:

*FEHLING reaction: Reductant/reducing sugar + CuSO4 + alkaline (a soluble base) → Cu2O (red
precipitation)

*BARFOED reaction: Reducing saccharide + Cu acetate [Cu(CH3COO)2] / CH3COOH → yellow to red

precipitation.

*BENEDICT reaction: Reducing saccharide + Na sulfate (Na2SO4) / Cu sulfate (CuSO4) → yellow to

red precipitation

*TOLLENS reaction: Reducing saccharide + AgNO3 / NH3 → silver mirror (Ag0)

b. Aromatic Nitro Compounds Reduction:

*PICRIC ACID reaction: Sugar solution + picric acid → aminonitrophenol (red)

c. Reactions based on the Formation of Furfural Derivatives:

*SELIWANOFF reaction (for ketoses): Furfural + resorcinol → red

*ANTRON reaction (for pentoses): Furfural + phloroglucinol → red

*MOLISCH reaction: Furfural + α-naphthol → purple-violet ring

*BIAL reaction (for pentoses): Furfural + orcinol → pink, violet

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 Fehling Test
 Sensitive test for detection of reducing sugars.

 Composed of two reagents; Fehling A (aq.copper sulphate) and Fehling B (aq. alkaline sodium
potassium tartarate).

 This test is used to distinguish aldehyde vs ketone functional groups where aldehyde gives
positive result by reducing Cu 2+ (Metal salt found in Fehling A) to Cu +1 in alkaline medium
(Fehling B) but ketones don't give positive test with it.

 Fehling's A is a blue aqueous solution of copper(II) sulfate crystals (generally dissolves in

water), while Fehling's B is a clear solution mixture of aqueous potassium sodium tartrate
(also known as Rochelle salt) and a strong alkali (commonly sodium hydroxide).

 When we are testing sugars we are using the same amount from both solutions ( 5 ml Fehling
A + 5 ml Fehling B + sample we are testing)

 Principle of Fehling Test:

 Aldehydes reduce the complexed copper(II) ion to copper(I) oxide. Because the solution is
alkaline, the aldehyde itself is oxidised to a salt of the corresponding carboxylic acid (carboxylate)

 When aldehydes are added to Fehling’s solution, they are easily oxidized by the cuprate (II)
complex to carboxylate (salt form of carboxylic acid). During this process, copper (II) ions get
reduced to copper (I) ions leaving a red precipitate of copper (I) oxide (Cu2O). The presence
of red precipitate indicates a positive result.

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 Figure 10: Principle of Fehling Test

Why Do Aldehydes And Ketones Behave Differently?

The difference between an aldehyde and a ketone is the presence of a hydrogen atom attached to the
carbon-oxygen double bond in the aldehyde. Ketones don't have that hydrogen.

The presence of that hydrogen atom makes aldehydes very easy to oxidise. Or, put another way, they
are strong reducing agents.

Because ketones don't have that particular hydrogen atom, they are resistant to oxidation

REMEMBER!

• Oxidation reaction (Releated to Oxygen or Hydrogen Transfer): Oxygen is gained or hydrogen

is lost.
• Reduction reaction (Releated to Oxygen or Hydrogen Transfer): Hydrogen is gained or
oxygen is lost.

OR in terms of Electron Transfer

• Oxidation is loss of electrons

• Reduction is gain of electrons
OIL RIG

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Figure 11: Oxidation / Reduction

In the above reaction, magnesium reduces the copper(II) ion by transferring electrons to the ion and
neutralizing its charge. Therefore, magnesium is a reducing agent. Another way of putting this is that
the copper(II) ion is removing electrons from the magnesium to create a magnesium ion. The copper(II)
ion is acting as an oxidizing agent.

What Is Formed When Aldehydes Are Oxidised?

It depends on whether the reaction is done under acidic or alkaline conditions. Under acidic conditions,
the aldehyde is oxidised to a carboxylic acid. Under alkaline conditions, this couldn't form because it
would react with the alkali, a salt is formed instead.

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During this process, the aldehyde is oxidized to carboxylic acid and the oxidizing agent is reduced.
Another way of framing this is to say that the aldehyde is the reducing agent in this process.

Figure 12 and 13: Oxidation of Aldehydes

* BUT Αlpha Hydroxy Ketone (Fructose) Also Gives Positive Result With Fehling Test, Why?

Fructose contain ketone as a reducing sugar. How this ketone functional group gives positive test
result with Fehling test as ketones do not get oxidized?

Alpha hydroxy ketone gets converted into enol by tautomerism (functional isomerism, migration of
atom, generally H) and then eventually turns into aldehyde group because of tautomerism. And that
aldehyde group gives positive test with Fehling’s solution.

Figure 14 and 15: From Alpha Hydroxy Ketone to Aldehyde

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 Molish Test
 This reagent is composed of α naphtol dissolved in ethanol.

 General test for all carbohydrates.

 The determination of a monosaccharide in a plant can be detected by MOLISCH reaction.

An appropriate and not very thin part of a plant to be examined must be cut. Above cross-
section of a plant, one drop of 15-20% α naphthol solution than some drops of concentrated
H2SO4 is added. If the cross-section of a plant suddenly turns into violet, then this indicates
the existence of saccahrides.

 This test is based on the dehydration of the carbohydrate by sulfuric acid or hydrochloric
acid to produce an aldehyde, which condenses with two molecules of a phenol, usually
alpha naphtol (dissolves in EtOH).

 When 5-C pentoses react with concentrated acid (H2SO4 or HCL) form furfural (OH groups of
monosacc. removed and become dehydrated). For 6-C hexoses 5 hydroxymethyl furfural
forms and both form of furfural reacts with alpha-naphtol and gives purple ring which this
ring is formation of triphenylmethane.

*Furfural: Dehydration, reacting monosaccharides with strong acid, monosaccharides reduce 3 water
units and forms furfural (especially pentoses give furfural).

Figure 16: Molisch test for 5 and 6 Carbon bearing carbohydrates

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 Seliwanoff Test
 This reagent composed of resorcinol + conc. acid.

 Used to distinguishes between aldose and ketose where ketoses react more rapidly then
aldoses. Ketoses form red color and aldoses give light red to pink color.

 Treatment with conc. Acid, ketoses are dehydrated more rapidly to give furfural derivatives
and on condensation with resorcinol give cherry red complex. The test will be answered by
fructose, sucrose and other keto containing carbohydrates.

 When monosacc. heated with Seliwanoff reagent ketoses forms furfural (ketoses + acid =
furfural formation) by reacting with conc.acid and resorcinol gives color changes.

Polysaccharide (Starch Determination)

Sample used: Wheat starch

Starch is composed of two fractions 1) amylose and 2) amylopectin. Amylose in starch is responsible
for the formation of a deep blue color in the presence of iodine.

REMEMBER; Polysaccharides, such as amylose or amylopectin, do have a hemiacetal group on one of

their terminal ends, but they are mainly nonreducing substances because there is only one reducing
group present for every 2,000–10,000 monosaccharidic units. In such a low concentration, the
reducing group does NOT give a positive test with Benedict’s or Fehling’s reagent.

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