REAC 446 modular • laboratory • program • in • chemistry program editor: H. A. Neidig Qualitative Testing for Carbohydrates prepared by James O. Schreck, University of Northern Colorado, and William M. Loffredo, East Stroudsburg University Purpose of the Experiment Study the reactions of various carbohydrates with reagents used to classify and identify these compounds. Background Information carbon atoms they contain. For example, we call monosaccharides containing 5 carbons, pentoses, The body gets its energy from three main classes of and those containing 6 carbon atoms, hexoses. Com- food: carbohydrates, proteins, and fats. Of these mon examples of monosaccharides include ribose, a classes, carbohydrates are the most important five-carbon aldose known as aldopentose; glucose source of energy. When digesting food, the body be- and galactose, six-carbon aldoses known as aldohex- gins to digest carbohydrates first. This process is rela- oses; and fructose, a ketohexose. tively efficient, producing waste products that are in- Carbohydrates containing a few monosaccharide nocuous (water) or readily removed (carbon dioxide). units are classified as oligosaccharides. Oligosac- Carbohydrates are widely distributed in plant tis- charides can be further subdivided into disaccharides sues and are even found in certain animal tissues, such (two-monosaccharide units), trisaccharides (three- as liver and muscle. Water-soluble carbohydrates often monosaccharide units), and so forth. Common exam- have a sweet taste and therefore are called sugars. ples of disaccharides are sucrose (glucose–fructose), Another term for carbohydrate is saccharide. table sugar; maltose (glucose–glucose), corn syrup; Carbohydrates are actually polyhydroxyalde- and lactose (galactose–glucose), milk sugar. hydes and polyhydroxyketones, often but not always Carbohydrates containing a large number of with the general formula (CH2O)n, where n equals 3 or monosaccharide units are classified as polysaccha- more. We refer to polyhydroxyaldehydes as aldoses rides. Of the many known polysaccharides, starch, and polyhydroxyketones as ketoses. cellulose, and glycogen are the most important. All Carbohydrates are divided into three general three are made up of hundreds of thousands of glu- classes, depending on the number of carbohydrate cose units connected in various patterns. One com- molecules they contain. Individual carbohydrate mole- mon characteristic of all oligo- and polysaccharides is cules are classified as monosaccharides. Mono- that they can be hydrolyzed to their monosaccharide saccharides are further classified by the number of units by heating in slightly acidic solution. Copyright © 1994 by Chemical Education Resources, Inc., P.O. Box 357, 220 S. Railroad, Palmyra, Pennsylvania 17078 No part of this laboratory program may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photo- copying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the United States of America 98 97 96 95 94 — 12 11 10987654321 2 REAC 446/Qualitative Testing for Carbohydrates CHO acidic H C OH dehydrating H CCH medium H C OH OHC C C H -3 H2O H C OH O furfural CH2OH ribose (a pentose) (Eq. 1) CHO H C OH acidic HOC H dehydrating H CCH H C OH medium HOCH2 C C CH -3 H2O HOHC O 5-hydroxymethylfurfural CH2OH glucose (a hexose) (Eq. 2) with furfural or 5-hydroxymethylfurfural to yield highly colored products. Reagents included in this class are Molisch reagent, Bial’s reagent, and Seliwanoff’s re- agent. These two classes of reagents are frequently Figure 1 Hemiacetal formation, showing the used together as part of a two-step analysis. hemiacetal structure in the dotted box and the two The third class of test reagents consists of solu- anomers tions containing copper(II) ions. These reagents oxi- dize certain carbohydrates. Two common reagents in Aldoses undergo an intramolecular reaction be- this class are Benedict’s and Barfoed’s reagents. We tween a hydroxyl group and the carbonyl group. The refer to the carbohydrates that reduce the copper(II) result is a cyclic hemiacetal, shown in Figure 1. We re- ions in these solutions to copper(I) oxide as reducing fer to the carbonyl carbon involved in the reaction as sugars. Reducing sugars are all aldoses containing the anomeric carbon (C* in Figure 1). At this carbon either a free aldehyde group or a cyclic hemiacetal. atom there are two stereochemical configurations pos- Note that under the reaction conditions used in tests sible, alpha (α) and beta (β). The two molecules con- with Benedict’s and Barfoed’s reagents, α-hydroxy- taining these configurations are called anomers. ketoses will also reduce copper(II) ions, giving a positive Cyclic hemiacetal anomers are more stable than (but incorrect) test result. This is due to the slow conver- α α the open-chain. Therefore, solutions of aldoses al- sion of the -hydroxyketone group to an -hydroxy- ways have a higher concentration of anomers than of aldehyde group, as shown in Figure 2. The R attached to the groups in Figure 2 represents the part of the mole- open-chain aldoses. A similar intramolecular reaction cule not involved in the reaction. occurs between a hydroxyl group and the carbonyl Disaccharides are oligosaccharides containing group of a ketose to form a hemiketal. two monosaccharide units joined by an acetal or ketal Carbohydrate test reagents can be divided into linkage. The linkage results from the reaction of the three general classes based on the type of reaction in- hemiacetal or hemiketal form of one monosaccharide volved. One class, dehydrating acids, causes carbo- hydrates to undergo dehydration, and form either O OH + - HO O furfural (from a pentose) as shown in Equation 1 or H or OH R CCH R CCH 5-hydroxymethylfurfural (from a hexose) as shown in H H Equation 2. The presence of furfural or 5-hydroxymethyl- α-hydroxyketone α-hydroxyaldehyde furfural can be detected by the addition of one of the second class of test reagents, called condensation Figure 2 Conversion of an α-hydroxyketone to reagents. These are phenolic compounds that react an α-hydroxyaldehyde REAC 446/Qualitative Testing for Carbohydrates 3 CH2OH CH2OH CH2OH CH2OH H C O H H C O H H C O H H C O H -H2O C H OH H C + C H OH H C C H OH H C C H OH H C +H2O HO CCOH HO CCOH HO CC O CCOH H OH H OH H OH H OH α-glucose α-glucose (a) maltose CH2OH CH2OH CH2OH CH2OH H C O H O H H C O H O H -H2O C H OH H C + C H OH C C H OH H C C H OH C +H2O HO CCOH HOC C HO CC O C C CH2OH CH2OH H OH OH H H OH OH H α-glucose β-fructose (b) sucrose Figure 3 (a) Formation of a reducing disaccharide; (b) formation of a nonreducing disaccharide with one of the hydroxyl groups on another mono- carbohydrates, so the test is used to distinguish be- saccharide. Disaccharides containing a free hemiacetal or tween carbohydrates and non-carbohydrates. The hemiketal, such as maltose in Figure 3(a), are reducing dehydration products of carbohydrates, furfural or sugars. Disaccharides containing no free hemiacetals 5-hydroxymethylfurfural, result from the reaction of or hemiketals, such as sucrose in Figure 3(b), are non- the sulfuric acid with pentoses and/or hexoses (Eq. reducing sugars. As shown in Figure 3, disaccharide 1 and 2). These products condense with α-naphthol linkages can be broken by the addition of water. This re- to yield a purple condensation product. The two-step action occurs slowly, but it can be catalyzed by acidic process is represented by Equation 3. media. carbohydrate → dehydration product → purple product (Eq. 3) Identifying Products of Carbohydrate Iodine and Potassium Iodide Test Condensation Reactions Starches form deeply colored blue-black com- Many carbohydrates can be identified using conden- plexes with iodine. Starches contain α-amylose, a heli- sation reagents, which react with the carbohydrates to cal saccharide polymer, and amylopectin. Iodine forms produce highly colored products. The precise nature of a large complex polysaccharide with the α-amylose the condensation reaction varies with the carbohy- helix, producing the blue-black color. Simpler oligo- drate under investigation. Often the carbohydrate is saccharides and monosaccharides do not form this initially dehydrated into smaller saccharide units, using complex with iodine. Thus, the I2/KI test can be used to a dehydrating acid. The smaller units, or molecules, distinguish starches from other carbohydrates. formed will produce a more highly colored complex Bial’s Test with the condensation reagent, as in Molisch’s, Bial’s, and Seliwanoff’s tests. In other cases the carbohy- Bial’s test uses concentrated hydrochloric acid as drate must possess certain structural features that al- the dehydrating acid and orcinol with a trace of iron(III) low it to form a condensation product, such as in the io- chloride as the condensation reagent. Bial’s test is used to distinguish between pentoses and hexoses. dine and potassium iodide test (I2/KI). The following section describes two types of condensation reac- Pentoses subjected to the test yield a blue or green tions: those used to generally classify carbohydrates, condensation product, while hexoses yield a muddy and those used to identify specific carbohydrates. brown-to-gray condensation product, as shown in Equations 4 and 5. Molisch test pentose → dehydration product: → The Molisch test uses concentrated sulfuric acid furfural as the dehydrating acid. This acid dehydrates all blue or green condensation product (Eq. 4) 4 REAC 446/Qualitative Testing for Carbohydrates hexose → dehydration product: → R – CHO 5-hydroxymethylfurfural 2+ → reducing +2Cu +2HO2 muddy brown-gray condensation product (Eq. 5) saccharide R – COOH Seliwanoff’s Test + carboxylic + Cu2 O(s, red) + 4H Seliwanoff’s test uses 6M hydrochloric acid as the acid (Eq.