Some sugars are capable of reducing silver ions to free silver, and copper(II) ions to copper(I) ions, under prescribed conditions. Such sugars are called reducing sugars. This reducing ability, which is useful in classifying sugars and in certain clinical tests, is dependent on the presence of (1) aldehydes, (2)
α− hydroxyketone groups such as in fructose, or (3) hemiacettal structures in cyclic molecules such as maltose. These groups are easily oxidized to carboxylic acid (or carboxylate ion) groups; the metal ions are thereby reduced. Several different reagents, including Tollens’,
Fehling’s, Benedict’s and Barfoed’s reagents, are used to detect reducing sugars. The Benedict,
Fehling and Barfoed tests depend on the formation of copper (I) oxide precipitate to indicate a positive reaction.
Reducing sugars are sugars that have the hemiactal or hemiketal functional group somewhere in their molecular structure. Either of these two functional groups will be in equilibrium with a free aldehyde group which will be very easily oxidized to a carboxylic acid. This oxidation will be accompanied by the reduction of the oxidizing agent often copper(II) ion or silver(I) ion. the copper(II) ion is reduced to copper(I) ion and the silver ion to silver metal. this is [was] a major way of silvering glass to make mirrors including reflecting telescope mirrors. It is the oxidation number of the oxidizing agent that is reduced in value.
In this test the presence of aldehydes but not ketones is detected by reduction of the deep blue solution of copper(II) to a red precipitate of insoluble copper oxide. The test is commonly used for reducing sugars but is known to be NOT specific for aldehydes. For example, fructose gives a positive test with Fehling's solution as does acetoin.
Two solutions are required:
A positive test is indicated by a green suspension and a red precipitate.
The test is sensitive enough that even 1 mg of glucose will produce the characteristic red colour of the compound.
Benedict's test determines whether a monosaccharide or disaccharide is a reducing sugar , and is hence similar in purpose to the Tollen’s and Fehling's test. This makes use of a single solution of copper(II) citrate which does not deteriorate as quickly on standing. Again Benedict’s solution contains copper sulphate. Reducing sugars reduce soluble bluer copper sulphate, containing copper(II) ions to insoluble red-brown copper oxide containing copper(I). The latter is seen as a precipitate.
To give a positive test, the carbohydrate must contain a hemiacetal which will hydrolyse in aqueous solution to the aldehyde form. Benedict's reagent is an alkaline solution containing Cu(II) ions, which oxidize the aldehyde to a carboxylic acid. In turn, the cupric ions are reduced to cuprous oxide, which forms a red precipitate.

RCHO + 2Cu
2+
+ 4OH
→
RCOOH + Cu
2
O + 2H
2
O
The colour ranges from green to yellow to orange to brick-red depending on the amount of reducing sugar in the sample; with a sample containing 1% glucose, the precipitate is usually brick-red. Benedicts's test will give a color change for any mono- or disaccharide containing a hemiacetal or hemiketal group. Since sucrose or table sugar does not contain these groups, it will not give a positive test.
This another variation of tests for reducing sugars, but determines if a carbohydrate is a monosaccharide or a disaccharide. Barfoed's reagent reacts with monosaccharides to produce cuprous oxide at a faster rate than disaccharides do:
RCHO + 2Cu
2+
+ 2H
2
O -----> RCOOH + Cu
2
O + 4H
+
A mixture of ethanoic (acetic) acid and copper(II) acetate, is added to the test solution and boiled.
If any reducing sugars are present a red precipitate of copper(I) oxide is formed within three minutes. The reaction will be negative in the presence of reducing disaccharide sugars as they are weaker reducing agents and react at a slower rate.

A copious amount of brick-red precipitate indicates a reducing monosaccharide. Some hydrolysis of disaccharides may lead to trace precipitates (disaccharides generally don't give any reaction even for ten minutes). The precipitate isn't nearly as voluminous as that seen with Benedict's test and tends to adhere to the walls of the test tube.
In the presence of conc. H
2
SO
4
sugars are dehydrated forming furfuryl derivates. Furfural is derived from the dehydration of pentoses and pentosans, while hydroxymethylfurfural is produced from hexoses and hexosans. Naphthol-(1) reacts with the cyclic aldehydes to form purple colored condensation products (furfuryl-diphenyl-methane-dyes) (1) . Although this test will detect compounds other than carbohydrates (i.e. glycoproteins), a negative result indicates the absence of carbohydrates.
Prepare Molisch's reagent by dissolving 0.5 g reagent grade
α
-naphthol in10 mL of 95% ethanol.
Store the reagent, protected from light, at room temperature. To test for carbohydrates, add 0.02 mL of the reagent to 1 mL of 0.1% carbohydrate (1 mg/mL) solution in a small test tube. After mixing, tilt the tube and carefully add without mixing, 0.5 mL of concentrated sulfuric acid by pouring it down the side of the tube. (Use a glass Pasteur pipette to add the H
2
SO
4
: do not use a mechanical pipettor with concentrated acids.) A red-violet layer at the interface between the acid
(bottom) and aqueous (upper) layers is a positive test for carbohydrates. (Reminder: Always add acid to water.)
Bial's Test (Pentoses)
Store the reagent protected from light. To test for pentoses, add 0.05 mL of 0.1% carbohydrate solution in water to 1 mL of Bial's reagent, (Use a glass Pasteur pipette to add the Bial's reagent: do not use a mechanical pipettor with concentrated acids.) and heat the solution in a boiling water bath for 2 minutes. A blue-green color indicates pentoses or nucleotides containing pentoses; a yellow-green color indicates hexoses, and disaccharides are yellow.
Bial's Test will distinguish between mono- and disaccharides, lactose will not react.

Store protected from light. To test for ketohexoses, add 0.1 mL of a 1% carbohydrate solution in water to 1 mL of the reagent, and heat the solution in a boiling water bath for 5 minutes. A deep red coloured precipitate within 5 minute indicates ketohexoses. Sucrose may give a positive ketohexose test because of partial hydrolysis to glucose and fructose. Other sugars give a red colour upon prolonged heating. Some sources say an apricot colour is negative. This depends on the concentration in the sample, and sugars like glucose give essentially no colour even after ten minutes.
A few drops of 0.01 M iodine in 0.12 M KI are added to a 1% solution of the carbohydrate in question. The immediate formation of a vivid blue color indicates amylose. With starch a a blueblack coloration forms due to the polyiodide complex formed.
Sugar Molisch Bial (
φ
)
2
NH G.O.
§
Resorcinol Benedict Barfoed Iodine
Ribose + (r/v) (bl/gr) - - - +(r ppt) + (r ppt) -
DNA* + (r/v) (y) (bl/gr) - - +(r ppt) + (r ppt) -
Fructose + (r/v) (y/gr) -
Galactose + (r/v) (y/gr) -
Glucose + (r/v) (y/gr) -
Sucrose + (r/v) (y) -
Lactose + (r/v) (y) -
-
-
+ (r)
-
+(br) -
- + (r)
- -
+(r ppt) + (r ppt) -
+(r ppt) + (r ppt) -
+(r ppt) + (r ppt) -
- - -
+(r ppt) - -
Maltose + (r/v) (y)
Amylose + (r/v) (y)
-
-
-
-
-
-
+(r ppt)
-
-
-
-
+ (bl)
Glycogen + (r/v) (y) - - - - - + (br)
+ = positive; - = negative; bl = blue ; br = brown ; gr = green ; r = red ; v = violet ; y = yellow; ppt = precipitate
* To test for DNA, the sample is first hydrolyzed in 10% trichloroacetic acid at 95
°
C for 10 minutes then diluted with two volumes of water before assaying with the diphenylamine test.
§
GO = glucose oxidase

Chemical Tests for Carbohydrates - Hydrolysis Test for Glucose
Disaccharides and polysaccharides can be hydrolyzed in acidic solution into their component monosaccharides, and then submitted to chemical tests like Benedict's test. In this experiment, several disaccharides and a sample of starch will be hydrolyzed, and tested for the presence of glucose. The glucose test will be carried out using a commercially available product called Tes-
Tape . Available at most drug stores, the tape contains the enzymes glucose oxidase and peroxidase, as well as ortho -toluidine. The glucose oxidase oxidizes glucose to gluconic acid and hydrogen peroxide. Once formed, the hydrogen peroxide reacts with peroxidase to produce oxygen, which oxidizes the ortho -toluidine to give green-coloured products.