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Reducing sugars

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.

Fehling's Test
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
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 + 2Cu2+ + 4OH- → RCOOH + Cu2O + 2H2O

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.

Barfoed's 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 + 2Cu2+ + 2H2O -----> RCOOH + Cu2O + 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.

Molisch Test
In the presence of conc. H2SO4 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 H2SO4: 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.
Resorcinol (Seliwanoff's) Test (Ketohexoses)
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.

Iodine Test (Starch/Amylose)

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 blue-
black coloration forms due to the polyiodide complex formed.

Qualitative Tests for Carbohydrates (resumo)

Sugar Molisch Bial (φ)2NH 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) - - + (r) +(r ppt) + (r ppt) -

Galactose + (r/v) (y/gr) - - - +(r ppt) + (r ppt) -

Glucose + (r/v) (y/gr) - +(br) - +(r ppt) + (r ppt) -

Sucrose + (r/v) (y) - - + (r) - - -

Lactose + (r/v) (y) - - - +(r ppt) - -

Maltose + (r/v) (y) - - - +(r ppt) - -

Amylose + (r/v) (y) - - - - - + (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

Este teste não sera feito na aula

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.