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Colorimetric Determination of Carbohydrate Unknowns

The identity of two chemical unknowns will be determined using a series of colorimetric chemical tests and carbohydrate standards. Introduction A colorimetric test is a chemical test in which a color is generated, or the color of a reagent changes, in response to a chemical reaction occurring with an analyte of interest. There are many different colorimetric tests for carbohydrates. Some of these colorimetric tests screen for specific carbohydrates, such as starch, while others test for broad classes of carbohydrates such as reducing sugars. In this lab we will focus on five colorimetric tests which, when used in sequence, will allow the positive identification of each of six different carbohydrates.

Table 1. Carbohydrates and their classifications.

Carbohydrate Arabinose Fructose Glucose Sucrose Lactose Starch

Classification monosaccharide aldopentose monosaccharide ketopentose monosaccharide aldohexose disaccharide (non-reducing) disaccharide (reducing) polysaccharide

The carbohydrates are divided into roughly three major groups: monosaccharides, disaccharides, and polysaccharides (Table 1). Within each group, the carbohydrates have unique chemical properties that will be demonstrated through the colorimetric tests.

Monosaccharides

H D-arabinose aldopentose HO H H

O H OH OH CH2OH OH HO O OH OH

-D-arabinofuranose

H H D-glucose aldohexose HO H H

O OH H OH OH CH2OH HO OH OH O OH OH

-D-glucopyranose

CH2OH O D-fructose ketohexose HO H H H OH OH CH2OH OH OH HO O OH OH

-D-fructofuranose

Figure 1. Open chain and hemiacetal/hemiketal ring structures of monosaccharides.

While there are three monosaccharides in this lab, each monosaccharide falls within a different subcategory which are illustrated in Figure 1. D-Arabinose is an aldopentose, Dglucose is an aldohexose, and D-fructose is a ketohexose. A series of related colorimetric tests based on the dehydration of carbohydrates is utilized to distinguish between these three monosaccharides.

Concentrated acid is used to dehydrate the carbohydrates. The product of the dehydration reaction depends on wether the carbohydrate is a pentose or hexose. Upon dehydration pentoses give furfural and hexoses yield 5-hydroxymethylfurfural (Figure 2.)

O carbohydrate pentose and hexose aldose and ketose Conc. Acid H O or H

O O

OH

furfural from pentose

5-hydroxymethylfurfural from hexose

Figure 2. Dehydration of carbohydrates to form furfural and 5-hydroxymehtylfurfural.

The aldehyde is then subsequently reacted with a compound called a phenol. The phenol reacts with the aldehyde to from a colored compound. The color of the compound (adduct) depends on the nature of the phenol used in the test. A general test for pentose and hexose carbohydrates utilizes -napthol and is called Molischs test (Figure 3).
O OH

Pentose or Hexose Carbohydrate

Conc. Acid H

O O R

2 (-naphthol) HO O R

Intermediate where R = H for pentose carbohydrate or R = CH2-OH for hexose carbohydrate

Adduct has purple color

Figure 3. Molischs test for carbohydrates utilizing strong acid and -naphthol.

While Molischs test is a general test for carbohydrates, tests for specific classes of carbohydrates have been developed based on the reaction shown in Molischs test. These tests utilize different phenols and slightly different conditions to react selectively with different types of carbohydrates. Bials test makes use of orcinol (Figure 4) which gives a blue-green adduct with pentoses and a green, brown or reddish-brown adduct with hexose. Seliwanoffs reagent produces a dark red adduct selectively with ketohexoses.
OH OH OH

H3C -napthol Molisch's Test produces purple adduct with carbohydrate orcinol

OH resorcinol

OH

Bial's Test produces blue-green adduct with pentose and green, brown reddish-brown with hexose

Seliwanoff's Test produces dark-red adduct with ketohexose

Figure 4. Comparison of the different phenols utilized, and observed selectivity, in the Molisch type colorimetric tests.

The two disaccharides in the lab are sucrose and lactose (Figure 5). Unlike monosaccharides, all disaccharides are not reducing sugars. A reducing sugar is one that contains a hemiacetal or hemiketal functional group such that the open chain form can be oxidized by either Tollens Reagent or Benedicts Solution. Differentiation of the two disaccharides will be accomplished using Tollens reagent.
HO HO OH HO OH OH O O OH OH OH OH O OH HO O OH O OH O

OH Lactose (1-4)glycoside reducing disaccharide

OH

Sucrose 1-2 diglycoside non-reducing disaccharide

Figure 5. Structures of the disaccharides lactose and sucrose.

Tollens reagent is a mild oxidizing agent of soluble silver ion known as ammonical silver nitrate. The silver (I) ion reacts to oxidize the aldehyde in the disaccharide to a carboxylate ion, and the silver (I) ion is reduced to metallic silver which precipitates on the sides of the container as a silver mirror, thus effecting a colorimetric determination (Figure 6).
HO OH HO OH OH O O OH OH HO OH OH O HO OH OH O OH 2 Ag(NH3)2OH HO OH OH O + 2 Ag + H2O + 3 NH3 OH oxidized disaccharide reduced silver HO OH OH O NH4 O OH

OH closed ring

OH open ring

Figure 6. The oxidation of lactose by Tollens Reagent.

The polysaccharide can be distinguished from the smaller di- and monosaccharides by its reaction with a solution of potassium iodide and iodine called Lugols solution. The iodide ion (I-) reacts with iodine (I2) to produce triiodide (I3 -). The triiodide ion complexes into the helical coils of the amylose (starch) producing a blue-black opaque complex (Figure 7). The smaller di- and monosaccharides do not have this helical structure of the amylose starch and do not complex with Lugols solution.

I I I I

Lugol's Solution amylose starch amylose starch triiodide ion complex

Figure 7. The starch/triiodide complex reaction.

The last test utilized is Fearons methylamine test for the disaccharides lactose and maltose. While maltose is not used in this lab, the test can be used to identify lactose. The test makes use of a basic solution of methylammonium chloride. The base reacts with the disaccharide causing hydrolysis to the monosaccharides and the creation of an enediol

functional group. The enediol complexes with the methylammonium ion to form a deep red complex. Using the reactions in sequence, a flowchart can be created that allows for the unique identification of each of the six carbohydrates (figure 8).

Arabinose/Fructose/Glucose/ Lactose/Sucrose/Starch Lugol's (I3-) Arabinose/Fructose/Glucose/ Lactose/Sucrose Tollen's Reagent Starch

Arabinose/Fructose/ Glucose/Lactose Seliwanoff

Sucrose

Arabinose/Glucose/ Lactose Bial

Fructose

Glucose/Lactose Methylamine

Arabinose

Lactose

Glucose

Figure 8. Flowchart showing the identification of carbohydrates using 5 different colorimetric tests.