Lecture 1.

WET METHODS OF
CARBOHYDRATE ANALYSES

Nomenclature of Carbohydrates
D, L Defines the configuration at C5
D has the OH at Right in Fischer projection
L has the OH at Left in Fischer projection
Gluco defines the configuration of the OH at C2, C4, C5. These
OHs are on same side while the C3-OH is opposite to others
, defines the configuration of the OH at C1, the anomeric carbon
Pyran indicates 6 member ring size
Furan indicates 5 member ring size

Examples follow

In Glucuronic acid C2, C4, C5 OHs are

on same side
H
C
H
HO
H
H

H
O
OH

C
H

O
OH

HO

OH

HO

OH

CO2H

glucuronic acid

OH
CO2H

galacturonic acid

Alditols
In Mannitol C2, C4,
C5 OHs are not at
same side in Fisher
Projection

CH2OH

CH2OH
HO
HO

H
H

OH

OH

OH

HO

OH
CH2OH

CH2OH

Mannitol

Xylitol

Conformations
Anomers
CH2OH
O OH
OH
OH

OH

OH

-D glucopyranose

[a] 25
D

+19o

CH2OH
O
OH
OH
OH

-D glucopyranose

+112o

Rotations of
Fresh
Solutions

25
o
[a]
For aged solutions
=
+52.7
D
Reason: Mutarotation is the best evidence for the cyclic
hemiacetal structure of D-(+)-glucose

Monosaccharides,Hemiacetal Formation II
CH2OH
H

H
OH

HO

CH2OH
O
..

OH

H
OH

OH

HO

O
OH
C
H

C5 OH attacks aldehyde giving a pyranose ring (6 member structure)

CH2OH

CH2OH

C H O H
..
C
C O
H
OH
H
C
C H

HO

OH

HO
H

C H O
C

OH
C

OH

OH

C4 OH attacks aldehyde giving a furanose ring (5 member structure)

-D glucofuranose
CH2OH
HO
O
OH

-D glucopyranose

Mutarotation

CH2OH
O
OH
OH
OH
OH

Ring closure between

OH C1 and C4 -OH
CHO

OH

CH2OH
OH
OH
CHO
OH
OH

CH2OH
O OH
HO
OH

H
HO
H
H

OH
H
OH
OH
CH2OH

D glucose

CH2OH
OH
CHO
OH
OH

OH

Ring closure between

C1 and C5 -OH

CH2OH
O OH
OH
OH

OH

-D glucofuranose

OH

-D glucopyranose

 Oligosaccharides
consist of several monosaccharide
residues joined together with glycosidic
linkages
di, tri, tetrasaccharides
(depending on the number of monosaccharides)

up to 10 - 20 monosaccharides (depending
on analytical techniques i.e GC vs LC/MS)

 Polysaccharides
refer to polymers composed of a large
number of monosaccharides linked by
glycosidic linkages
ex. Cellulose
Cellobiose
CH2OH
HO
HO

OH

CH2OH

OH
HO
O

O
CH2OH

anhydroglucopyranose
unit

O
HO

OH

OH
HO
O

OH
O
CH2OH

n = 1 -5000
oxygen bridge
(ether-type or
glycosidic bond)

Cellulose
-D-anhydroglucopyranose units
linked by (1,4)-glycosidic bonds
6

HO
HO

OH
CH2OH

Non-Reducing
End-Group

CH2OH

O
HO

5
3

HO
O

OH

OH

3'

O
1

4'

5'

2'

CH2OH O

6'

CH2OH

1'

O
HO
n

OH
OH

Reducing
End-Group
(potential aldehyde)

Polysaccharides
Polysaccharides can be divided into two classes
Homopolysaccharides
consist of only one kind of monosaccharide
ex cellulose

Heteropolysaccharides
consist of two or more kinds of
monosaccharides
ex galactoglucomannans

Polysaccharides
Polysaccharides can not only have
different sequences of monosaccharide
units, but also different sequences of
glycosidic linkages and different kinds of
branching
a very high degree of diversity for
polysaccharides and their structurefunction relationships

Plant Polysaccharides
The conformation of individual
monosaccharide residues in a polysaccharide
is relatively fixed, however, joined by
glycosidic linkages, they can rotate to give
different chain conformations.
1,4 glycosidic
linkage

OH
O
HO

O
HO

O
HO

OH

1,6 glycosidic
linkage

HO
HO

HO

O
HO

OH

HO
HO

HO

Plant Polysaccharides
The different kinds of primary structures
that result in secondary and tertiary
structures give different kinds of
properties
water solubility, aggregation and
crystallization, viscosity, gelation, etc.

Polysaccharides have a variety of

functions
Storage of chemical energy in
photosynthesis

Starch
Starch is composed completely of Dglucose
found in the leaves, stems, roots,
seeds etc in higher plants
stores the chemical energy produced
by photosynthesis
Most starches are composed of two types
of polysaccharides - amylose and
amylopectin
amylose - a mixture of linear
polysaccharides of D-glucose units
linked -(1-4) to each other

Starch Polymer Components

Amylose

Amylopectin (1 residue in every 20 is 16 linked to branch off)

The Components of Starch

Amylose
O
HO

Amylopectin

OH
O

OH O

OH
O
HO

OHO

OH
O

OH
O
(1-4)
HO
HO

OH O
OH O HO

OH
O

O HO

OHO
HO
(1-4)

OH
O (1-6)
O
OH
O
HO

OH
O

O
OH
HO
O

OH O
HO
OH O

OH
O

HO
O

OH
O

Starch tertiary structure (Helix)

QUALITATIVE ANALYSIS

There various tests that can be used to

detect
the presence or absence of
carbohydrates or sugars. Some of these
are:
Molisch Reaction
Anthrone Reaction
Iodine Test
Benedict Test

MOLISCH REACTION
In this reaction the furfural that is formed from
the carbohydrate by the sulfuric acid
condenses with the phenol to give the
characteristic color.
PROCEDURE
Two ml of a sample solution is placed in a
test tube. Two drops of the Molisch reagent
(a solution of -napthol in 95% ethanol) is
added. The solution is then poured slowly
into a tube containing two ml of concentrated
sulfuric acid so that two layers form.

MOLISCH REACTION
A positive test is indicated by the formation
of a purple product at the interface of the
two layers.

a negative test (left) and a positive test

(right)

ANTHRONE REACTION
Anthrone, 9,10-dihydro-9-ketoanthracene
reacts with many carbohydrates to give a
green color.
PROCEDURE
1 ml of a sample solution is placed in a test
tube. 2ml of a 0.2% of Anthrone in
conconcentrated sulfuric acid is added. In the
presence of carbohydrates a clear green
color will appear and will rapidly increase in
intensity until a dark blue-green solution
results.

QUANTITATIVE ANALYSIS
The quantitative methods for the
estimation of sugars and carbohydrates
depend on the properties of reduction and
optical rotation that the sugars have.
Some of the quantitative methods used
are;
Munson and Walker Method
Iodide-Thiosulfate Method
Lane-Eynon Titrimetric Method

LANE-EYNON METHOD
This is a short and rapid method and often
the most accurate method for the
estimation of reducing sugars. It is based
on a determination of the volume of a test
solution required required to reduce
completely a known volume of alkaline
copper reagent. The end point is indicated
by the use of an internal indicator,
methylene blue.

LANE-EYNON METHOD

SAMPLE PREPARATION
12.5g of the sample is dissolved in water.
25ml of 10% neutral lead acetate solution is
added.
Some alumina cream is added and made up
to 250ml in a volumetric flask.
The solution is shaken thoroughly and
filtered.
10ml 10% solution of potassium oxalate is to
100ml of the filtrate and made up to 500ml,
shaken and filtered

LANE-EYNON METHOD

PROCEDURE
10ml of the mixed Fehling reagent is placed in a 250ml
Erlenmeyer flask.
The sugar solution is transferred into a burette and
suspended over the Erlenmeyer flask.
15ml of the sugar solution is added to the flask and heated to
boiling.
The solution is boiled for about 15 seconds and portions of
the sugar solution is added rapidly until only the faintest
perceptible blue color remains.
2-5 drops of a 1% aqueous solution of methylene blue is
added and heating is continued.
The sugar solution is added dropwise until the titrtion is
complete which is shown by the reduction of the dye.

LANE-EYNON METHOD
The amount of sugar may be calculated by
the formula;

The factor is obtained in Literature, in

which the factor for each titration from 15
to 50ml is given.