Reactions of Carbohydrates1

Animals largely depend on plant source to obtain carbohydrates though they can synthesize carbohydrates from non carbohydrates sources like glycerol and amino acids in their body (gluconeogenesis).

The glucose is the major form of carbohydrate absorbed from the gut in humans.

According to the metabolic status it has different fates–

The classification of carbohydrates will be useful for the detection of various types of carbohydrates by different chemical tests.

Proteoglycans, e.g. Heparin (D-glucosamine sulfate + D-sulfated iduronic acid)

Hyaluronic acid (D-β glucuronic acid + N-acetylglucosamine).

The reactions described below, are applied in the identification of sugars.

Observation: A reddish violet ring appears at the junction of two liquids.

Inference: Indicates presence of a carbohydrate and hence the presence of monosaccharide.

Principle: Concentrated acid dehydrates the sugar to form furfural (in the case of pentoses) or furfural derivatives (hexoses and heptoses) which then condense with α-naphthol to give a reddish violet colored complex

Application of the test: Used as a general test to detect carbohydrates.

Observation: The entire body of the solution will be filled with a precipitate, the color of which varies with the concentration of the sugar solution—green, yellow, orange or red.

In the absence of reducing substance, blue color of the Benedict's reagent remains as such. The test is sensitive up to 0.1-0.15 gm% of sugar solution (that is Benedict's test will not be 6positive with solutions containing less than 0.1-0.15 gm% of sugar).

Inference: Reducing monosaccharides, glucose, fructose, galactose and mannose give a positive reaction with Benedict's reagent.

The color of the precipitate give an idea about the concentration of the sugar solution as shown below.

Thus, Benedict's test is described as a semi-quantitative test.

Principle: (see Fig. 1A-4) Carbohydrates with a free aldehyde or keto group have the ability to reduce various metallic ions. In this test cupric ions are reduced to cuprous ions by the enediols formed from sugars in the alkaline medium of Benedict's reagent.

Benedict's reagent contains copper sulphate, sodium citrate and sodium carbonate.

Copper sulphate dissociate to give sufficient cupric ions (in the form of cupric hydroxide) for the reduction reactions to occur.

Sodium citrate keeps the cupric hydroxide in solution without getting precipitated.

Sodium carbonate (Na₂CO₃) make the pH of the medium alkaline.

In the alkaline medium sugars form enediols which are powerful reducing agents. They reduce blue cupric hydroxide to insoluble yellow to red cuprous oxide.

Application of the test: To detect reducing sugars. It is widely used in detecting glucose in urine even though not specific for glucose.

Observation: A red precipitate clinging to the bottom most part of the test tube forms, in the presence of a monosaccharide.

Inference: The test is answered by monosaccharides only, e.g. glucose, fructose, galactose, mannose.

Principle: It is a reduction test. Reducing property owes to the carbonyl group (aldehyde or keto group). Barfoed's reagent is copper acetate in acetic acid.

Difference between Barfoed's test and Benedict's test: Barfoed's test differs from Benedict's test with respect to the pH of the medium. It is alkaline in the case of Benedict's and acidic in the case of Barfoed's test. In the acid medium monosaccharides enolize much more readily than disaccharides and these enediols reduce cupric ions released by copper acetate of Barfoed's reagent to produce a red precipitate.

Chemistry of the test: Reduction reaction as shown under Benedict's test.

Observation: Positive reaction is indicated by the development of violet color (Fig. 1A-6).

Inference: Development of violet color within 30 seconds of boiling indicates presence of a keto sugar, e.g. fructose.

Principle: A dehydration reaction which owe to the hydroxyl groups of the sugar. Concentrated HCl being weaker than concentrated sulphuric acid, dehydrate ketoses (e.g. fructose) more readily than aldoses to form hydroxymethyl furfural, which then condenses with α-naphthol to form a violet colored complex.

Chemistry of the test: Dehydration reaction as shown under Molisch test.

Aberrant reaction: If red color develops instead of violet color due to charring action of acid, dilute the sugar sample with water and conduct the test with diluted sugar solution (Fig. 1A-7).

Observation: Positive reaction gives a red color within half a minute (Fig. 1A-8).

Inference: This test is given by ketoses. e.g. fructose.

Principle: A dehydration reaction due to the hydroxyl groups of the sugar. Selivanoff's reagent is resorcinol in dilute hydrochloric acid. Ketoses (e.g. fructose) are more readily dehydrated by HCl than the aldoses to form hydroxymethyl furfural which then condenses with resorcinol of Seliwanoff's reagent to form a red colored complex.

Avoid showing under the tap water because rapid cooling disturbs crystallization where as slow cooling ensures crystallization (ideally within the water bath itself).

Observation: Crystals are formed readily (within 1–5 minutes) at the room temperature in the case of mannose. For other sugars minimum time required in minutes in the water bath for the formation of insoluble yellow osazone is given in the Table 1A-2.

Look under the microscope to view the crystals (see Fig. 1A-9).

Inference: Glucose, fructose, mannose yield the same shaped phenyl osazone crystals because of the elimination of differences in configuration about the carbon atoms 1 and 2 during osazone formation.

Principle: The reaction involves the carbonyl carbon (either aldehyde or ketone as the case may be) and the adjacent carbon. One molecule of sugar reacts with one molecule of phenyl-hydrazine to form phenylhydrazone which then reacts with two additional phenyl hydrazine molecules to form the osazones as shown in the figure 1A-10.

Maltose (α-D-glucopyranosyl-(1→4) α-D-glucopyranose) (Fig. 1B-1): Maltose yield 2 glucose molecules upon hydrolysis. Maltose is formed from the hydrolysis of starch by the action of the enzyme maltase. It is also produced as an intermediate product of mineral acid hydrolysis of starch. It is dextrorotatory, exhibits mutarotation, reduces metallic ions in alkaline solutions. Like other disaccharides maltose is hydrolyzed by dilute acid leading to the formation of two molecules of glucose. With phenyl hydrazine maltose forms maltosazone.

Examples for other disaccharides that produce only glucose upon hydrolysis:

Sucrose (α-D-glucopyranosyl-β-D fructofuranoside): (see Fig. 1B-3).

Hydrolysis of sucrose yields one molecule of glucose and one molecule of fructose. Sucrose is dextrorotatory. After hydrolysis by enzymes or weak acids, it becomes levorotatory. This is because of the formation of fructose upon hydrolysis, which is strongly levorotatory than the glucose. Thus the change of optical rotation of sucrose solution from dextro to levo rotation upon hydrolysis is known as inversion and the mixture of glucose and fructose obtained is called invert sugar.

But prolonged boiling with phenylhydrazine in acid medium will form osazone due to the reaction of products of hydrolysis of sucrose with phenylhydrazine and not due to the reaction of intact sucrose molecules with phenylhydrazine.

Application: Useful to differentiate monosaccharides from disaccharides.

Lactose gives a characteristic yellow puff shaped lactosazone crystals (see Fig. 1B-4).

Maltose: Individual crystals of maltosazone looks like a yellow colored petal and when grouped looks like a sun flower (see Fig. 1B-5).

Application: Useful to differentiate disaccharides.

Observation: Sucrose gives bright red color (see fig. 1A-8) whereas lactose and maltose do not give red color.

Inference: Sucrose upon acid hydrolysis by the HCl in the Seliwanoff's reagent yields a keto sugar, fructose. Fructose being a keto sugar gives positive response to Seliwanoff's test as described under monosaccharides. Whereas lactose (galactose + glucose) and maltose (glucose + glucose) contain no keto sugar and cannot give positive response to this test upon acid hydrolysis by the HCl present in the Seliwanoff's reagent.

Principle: The disaccharide sucrose contains glucose and fructose. Fructose formed from sucrose upon acid hydrolysis by the HCl of Seliwanoff's reagent, is dehydrated by the acid HCl to form hydroxymethyl furfural which then condenses with the resorcinol of Seliwanoff's reagent to form a red colored complex.

Observation: Sucrose gives violet color (see Fig. 1A-6) whereas lactose and maltose do not give violet color.

Inference: Sucrose upon acid hydrolysis by the HCl added in the test yields a keto sugar fructose. Fructose being a keto sugar gives positive response to Rapid furfural test as described under monosaccharides. Where as lactose (galactose + glucose) and maltose (glucose + glucose) contain no keto sugar and cannot give positive response to this test.

Principle: The disaccharide sucrose contains glucose and fructose. Fructose formed from sucrose upon acid hydrolysis by the HCl, is dehydrated by the same HCl to form hydroxymethyl furfural which then condenses with the α-naphthol of Molisch reagent to form a violet colored complex.

Observation: Unboiled sucrose solution will not give a positive response to Benedict's test where as boiled portion gives a positive response.

Inference: Sucrose is hydrolyzed by HCl in the first step to form glucose and fructose and the medium is neutralized by the 2% sodium carbonate.

In the second step, products of acid hydrolysis reduce cupric ions to red cuprous oxide.

Thymol blue indicator contains two components that work at acid range (pH range 1.2–2.8; color change – red to yellow) and at alkaline range (pH range 8.0–9.6; color change – yellow to blue).

In all types the linkage between the monosaccharide units is the glycosidic bond.

This may be α or β which join the respective units through 1 → 2, 1 → 3, 1 → 4 or 1 → 4 linkages in the linear sequence or at branch points in the polymer.

Polysaccharides are classified based on the type of monosaccharide units present in them.

We will discuss the reactions of starch in this chapter in order to understand the chemical properties of polysaccharides in general.

Starch → Soluble starch → Amylodextrins → Erythrodextrins → Achrodextrins → Maltose → Glucose. When the hydrolytic stage reaches to the level of formation of maltose and glucose iodine test becomes negative and Benedict's test becomes positive.