Isolation and Characterization of Carbohydrates
BACKGROUND INFORMATION
The most important sources of energy, carbohydrates are polyhydroxyaldehydes
(aldoses) and polyhydroxyketones (ketoses) with the general formula (CH₂O), where n equals 3
or more. They are widely distributed in plants and even found in certain animal tissues such as
the liver and muscles. Carbohydrates are divided into three general classes depending on the
number of monosaccharide units they contain,
1. Monosaccharides - simple sugars that cannot be hydrolyzed
2. Oligosaccharides - contain 2-10 monosaccharide units
3. Polysaccharides - contain more than 10 monosaccharide units
Monosaccharides or simple sugars are further classified by the number of carbon atoms
they contain. Monosaccharides containing five carbons are called pentoses while those
containing 6 carbon atoms are known as hexoses. Ribose and xylose are common examples of
aldopentoses while glucose and galactose are aldohexoses Meanwhile, fructose is an example
of a ketohexose. Oligosaccharides are subdivided into disaccharides (2-monosaccharide units),
trisaccharides (3-monosaccharide units), and so forth. Common examples of disaccharides are
sucrose, maltose, and lactose. Polysaccharides can be made up of hundreds of thousands of
monosaccharide units connected in various patterns. Of the many known polysaccharides,
starch, cellulose, and glycogen are the most important homopolysaccharides
A common characteristic of oligosaccharides and polysaccharides is that they can be hydrolyzed
by being heated in a slightly acidic solution. Carbohydrate test reagents can be divided into two
general classes based on the type of reaction involved. The first class is a 2-step analysis
consisting of the use of dehydrating acids followed by condensation reagents. Dehydrating
acids convert pentoses into furfural and hexoses into 5-hydroxymethyl furfural which then
reacts with phenolic compounds to yield highly colored products. This is the principle involved
in the Molisch's, Anthrone, Bial's, and Seliwanoff's tests. The second class of test reagents uses
solutions containing copper (II) ions. The carbohydrate reduces the copper (II) ions into copper
(1) oxide. Reducing sugars include all aldoses containing either a free aldehyde group or a cyclic
hemiacetal.
Qualitative Tests for Carbohydrates
Benedict's Test. Reducing sugars are usually detected using Benedict's reagent which contains
copper (II) ions in an alkaline solution with sodium citrate to keep the cupric ions in solution.
The alkaline condition of this test causes the isomeric transformation of ketoses to aldoses,
resulting in the reduction of blue cupric ion to cuprous oxide (Cu,O), a brick-red precipitate, by
all monosaccharides and most disaccharides.
RCHO + 2Cu + 5OH → RCOO + CuO + 3H,O
Reducing carbohydrates
Carboxylate
Barfoed's Test. Barfoed's solution contains cupric ions in an acidic medium. The milder
condition oxidizes the monosaccharides but not the disaccharides. If the time of heating is
controlled, disaccharides will not react, but reducing monosaccharides will produce the positive
brick-red Cu₂O↓ result. Ketoses do not isomerize with this reagent.
Seliwanoff's Test. Seliwanoff's reagent is resorcinol in 6 M HCI. Ketohexoses undergo
dehydration to form a hydroxymethylfurfural that condenses with resorcinol to form a cherryred condensation product. Other sugars may produce yellow to pink colors.
Bial's-Orcinol Test. The reagent contains orcinol (5-methylresorcinol) in concentrated HCl with a
small amount of FeCl₁. Pentoses undergo dehydration to form furfural that condenses with
orcinol to create a blue-green solution.
Mucic Acid Test. Aldohexoses are converted to their corresponding dicarboxylic acids in the
presence of strong oxidizing agents like concentrated HNO, The dicarboxyle acid (music acid)
produced from the oxidation of galactose is relatively insoluble and separates out as colorless
crystals.
Phenylhydrazone Test. Reducing sugars may be differentiated from each other by the
phenylhydrazones (osazones) they form with a phenylhydrazine reagent. The oxazones from
different reducing sugars have the characteristic crystalline forms and a definite time of
formation. They also differ in their relative solubility in hot water A microscopic examination
and comparison of the formed osazones with reference standards may lead to the identification
of the reducing sugar.
Thin-layer Chromatography
Thin-layer chromatography is based on a phenomenon called adsorption. It provides a better
resolution and faster development than paper chromatography in the separation and
identification of unknowns. The partial characterization and tentative identification of an
unknown can be carried out by comparing its Rf value to that of the standard using a particular
solvent system.
Solvent systems:
a. n-butyl alcohol: acetic acid: ether: water (9:6:3:1~v/v)
b. acetonitrile: water (85:15~v/v)
c. acetonitrile: ethyl acetate: n-propyl alcohol: water (85:20:50:90~v/v)
Quantitative Analysis
The amount of carbohydrates present in a given sample can be measured by Nelson's method
which is based on the capacity of the free reducing groups of sugars in a carbohydrate sample
to reduce Cu in an alkaline solution. This method is sensitive and the data obtained are
reproducible.
In this determination, the amount of free reducing sugars in the sample is measured
colorimetrically and directly related to the molybdenum blue formed via a series of
oxidation/reduction reactions.