QUALITATIVE ANALYSIS OF CARBOHYDRATES

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Objective

; To use analytical chemical techniques to characterize carbohydrates.

Introduction

    Biological samples are composed of four different types of macromolecules: carbohydrates, lipids, nucleic acids, and proteins. In this laboratory, we will concentrate on carbohydrates.

    Carbohydrates are polymers of sugar molecules that serve as storage molecules or as structural molecules. Starch and glycogen are examples of storage molecules. Hydrolysis of either starch or glycogen releases the monosaccharide glucose, which may then serve as fuel for cellular work or as a carbon source for synthesis of other organic molecules. Cellulose, which makes plant cell walls tough, and chitin, the main ingredient of an insect’s exoskeleton, are examples of structural molecules.

    In order to identify what types of carbohydrates are present in a solution, various chemical assays can be employed.

Anthrone Test

    The anthrone test can be used for the qualitative and quantitative estimation of polysaccharides as well as monosaccharides. The test is based on the dehydration of monosaccharides to furfural derivatives, e.g. hydroxymethylfurfural. Furfural derivatives react with anthrone to form a deep green color.

    OOHCOHCH2

     Hydroxymethylfurfural

    O

     Anthrone

Benedict’s Test

    The fact that aldehyde groups and ketone groups may participate in chemical reactions is the basis for the Benedicts’s test. In this procedure, Benedict’s reagent, which contains a basic

    solution of cupric ions, is mixed with a sugar. The copper ions will attract the electrons from the aldehyde or ketone group changing the charged copper ions to a neutral metal. Copper ions are blue in color whereas copper metal is orange. A disaccharide, a sugar composed of two monosaccharides, will react with the Benedict’s reagent only if it contains an exposed aldehyde or ketone group. This type of sugar is known as a reducing sugar.

Barfoed’s Test

    The Barfoed’s test is used to distinguish between reducing monosaccharides and disaccharides. Barfoed’s reagent contains cupric ions in an acidic solution. Milder conditions and careful control of the heating time allow monosaccharides, but not disaccharides, to be oxidized. The cupric ions are reduced in the presence of a monosaccharide resulting in a red precipitate.

Seliwanoff’s Test

    Seliwanoff’s test is used to distinguish between ketoses and aldoses. Seliwanoff’s test contains resorcinol in 6M hydrochloric acid. Ketoses are more rapidly dehydrated than aldoses when heated. The dehydrated ketose, hydroxymethyl furfural, then reacts with the resorcinol to produce a deep cherry red color. Aldoses may react slightly to produce a faint pink color.

    OH

    OH

    Resorcinol

    Bial’s Test

Bial’s reagent contains orcinol in concentrated HCl with a small amount of FeCl catalyst. 3

    Pentoses react with Bial’s reagent to form furfural, which condenses with orcinol to produce a blue-green product.

    OHO

    O

    OH

    orcinolfurfural

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Iodine Test

Iodine forms colored complexes with polysaccharides. The color of the complex depends on the

    3-D structure of the polysaccharide. Starch is a coiled structure, which turns blue when bound to

    IKI whereas glycogen, which is a branched molecule, turns red-violet.

Method

    The following compounds will be used for each test: starch, glucose, fructose, ribose, maltose,

    sucrose, and water (control).

A. Anthrone Test for Carbohydrates

    1. Place 1 ml of each sample to separate test tubes.

    2. Add 5 ml of anthrone reagent.

    3. Mix and place tubes in boiling water bath for 10 min.

B. Benedict’s Test for Reducing Carbohydrates

    1. Add 5 drops of each sample to separate test tubes.

    2. Add 2 ml of Benedict’s reagent.

    3. Mix and place tubes in boiling water bath for 5 min.

C. Barfoed’s Test for Reducing Monosaccharides

    1. Add 1 ml of each sample to separate test tubes.

    2. Add 2 ml of Barfoed’s Reagent.

    3. Mix and place tubes in boiling water bath for 1 min.

D. Seliwanoff’s Test for Ketoses

    1. Add 2 ml of Seliwanoff’s reagent to seven test tubes.

    2. Place 0.05 ml of each test sample in separate test tubes.

    3. Mix and place tubes in boiling water bath for 1 min.

E. Bial’s Test for Pentoses

    1. Place 1 ml of each test sample in a separate test tube.

    2. Add 2.5 ml of Bial’s reagent.

    3. Mix and place tubes in boiling water bath for 1 min.

    4. Allow tubes to cool to room temperature.

    5. Add 3 ml of amyl alcohol. You should observe two distinct phases at this time.

    6. Record which compounds give a rapid positive result (blue-green color).

F. Iodine Test for Starch

    1. Add 2 ml of each sample in separate test tubes.

    2. Add four drops of IKI solution.

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